IL326291A - Method for improving gelation properties of alternative proteins - Google Patents

Description

Method of improving gelation properties of alternative proteins Technical Field The present invention is directed to methods for providing an altered and favorablyimproved gelation pattern of plant-based protein ingredients suitable for use alternativeconsumables, including vegetarian or vegan meat or dairy analogues. The proteiningredients have desirable organoleptic and viscoelastic properties stemming from 5improved methodologies in plant genetics.

Background In view of an increasing consumer awareness regarding animal welfare and diet-associatedillnesses, including obesity, type II diabetes, prevention of dementia and cardiovasculardiseases, the development of healthier food products is of increasing interest and there is 10an ever-increasing consumer demand for plant-based food and food additives. In view ofchallenges related to climate change and a steady increase in the world population furtherincrease the awareness related to ecological sustainability in the context of developing newseeds, planting and using the same.

Producers are thus facing the challenges of providing plant-based alternatives at scale that 15meet consumers’ expectations in taste, texture, and other functional and organolepticproperties.

To meet these demands, several sources of plant-based proteins have been consideredby producers, including but not limited to: soybeans; peas, chickpeas and split peas;cranberries; beans such as navy, pinto, adzuki, fava, lima, black, red kidney, and mung 20beans; pumpkin seed and seed from other squash; and grains such as rice, sorghum, andmillet. Consumers look for clean-labelled, locally-sourced and sustainable ingredients, which factors are also considered by producers. Further, certain consumers do not wish toconsume animal products, including milk from cows, at all in view of its animal origin, dueto lactose intolerance, or due to dairy allergies and the like. They may also see potentialenvironmental sustainability issues. The global consumable industry has a continuing needto find plant-based protein sources that meet these requirements and preferences. 5 Peas (Pisum sativum or Pisum sativum L.) are annual plants with a one-year life cyclebelonging to the family of Leguminosae. Peas belong to the oldest cultivated crops anddomestication of wild pea plants likely already began with the start of agriculture about11,000 years ago. Pea plants contain symbiotic bacteria within their root system with thespecial ability to fix nitrogen from the atmosphere, making them a highly attractive source 10of protein which meets the preferences of locally grown, sustainable agricultural practices.

One major challenge of working with pea protein ingredients and isolates or concentratesand the like thereof as part of compositions in consumable applications has to do withinconsistent functional properties of commercially available pea proteins, especiallyviscoelastic properties such as gelation. Indeed, pea protein gelation differs significantly 15from the process known from whey protein and pea protein aggregation under thermaland/or mechanical treatment often leads to undesired aggregation. Only subtly variationsin protein content can result in poor textural performance, melting profiles, structuralintegrity, and the like, making the use of pea proteins as a versatile plant-based proteinsource difficult at date. 20 The core storage proteins of peas can be categorized as globulins and albumins. Globulinsrepresent the predominant fraction of pea proteins (~ 50 to 80% of total protein) and aredivided into legumin, vicilin, and convicilin proteins. Legumin is a hexamer composed ofacidic and basic subunits connected by disulfide bonds (-S-S-). Vicilin usually occurs as atrimer with monomers of 50 kDa each (Liang and Tang, 2013, 25https://doi.org/10.1016/j.foodhyd.2013.04.005). The protein is known to fragment into 33kDa (αβ), 30 kDa (βγ), 19 kDa (α), 13.5 kDa ( β), and 16 or 12.5 kDa ( γ) subunits in a(denaturing) SDS-PAGE (O'Kane et al., 2004, https://doi.org/10.1021/jf035215h). Incontrast to legumin, vicilin is less hydrophobic. Structurally, vicilin does not contain anydisulphide bonds. However, dependent on genotype, vicilin may contain one or two 30cysteine groups, which show a potential to form covalent bonds during aggregation (Chihiet al., 2016, https://doi.org/10.1021/acs.jafc.6b00087). The third major globulin protein isconvicilin, which shows high homology to vicilin. It forms tetramers of 290 kDa withmonomers of ~ 70 kDa. The amino acid sequence building up a monomer contains onecysteine group (Shrestha et al., 2023, https://doi.org/10.1016/j.foodchem.2023.135464). 35 Regarding the food-relevant properties, it was described that vicilin protein fractions canundergo heat-induced gelation, whereas convicilin has a negative effect on gelation(O’Kane et al. 2004, supra). Legumin has a better emulsion capacity than vicilin (Cserhalmiet al., 1998, Emulsifying properties, surface hydrophobicity and thermal denaturation ofpea protein fractions. Acta Alimentaria, 27, 357–363). Albumin has the best emulsifying 5properties but does not exhibit effective gelation (Shanthakumar et al., 2022,https://doi.org/10.3390/molecules27165354). At date, however, there is no detailedinformation or a suitable pea line available that has a high gelation pattern. Such a pea,and methods of producing suitable pea protein compositions thereof, is of great interest forthe food and cosmetic market, as these compositions can be used to prepare, inter alia, a 10meat of fish like texture with a certain viscoelastic stability that is lacking in reference peaingredients in view of the anti-gelation properties of convicilin.

As a drawback for food production, all globulin proteins all have a strong tendency foraggregation, as the processing after harvest, including thermal and mechanical treatments,results in denaturation and primary and subsequent aggregation. This hampers the control 15of the gelation pattern. Pea protein synthesis in the plant is regulated by complex geneticmechanisms involving multigene families encoding various protein types, and themechanisms also reflect the developmental and metabolic processes that occur during thelifecycle of the pea plant. Gelation is the most crucial characteristic of globular protein. Forfood production using pea proteins, it is of utmost importance to be in a position to regulate 20the globulin content, as the globulin content significantly influences the behavior of the finalproduct. This is complicated in case the raw materials contain a varying composition ofglobulins, including convicilin, naturally varies. Further, a pea protein compositions for usein a liquid or in a solid products is inherently associated with different needs.

In 2019, a pea reference genome was published, describing a total of 40 seed protein 25storage genes along with 210 upstream regulatory motifs for these genes (Kreplak, et al.,Nat. Genet. 51, 2019, https://doi.org/10.1038/s41588-019-0480-1), indicating that theunderlying genetic framework controlling pea protein content is large and complex. Thefunctional properties of individual storage proteins such as globulins and albumin have alsobeen studied and published. However, at date no correlation has thus far been made 30between the complex genetic framework of the pea plant and the functional properties ofpea proteins produced in pea plant.

Therefore, there is currently a great need in providing homogeneously and stable peaprotein ingredients, isolates, flours and compositions showing a uniform and predictablegelation pattern, wherein, for food safety and sustainability purposes, the identification and 35 generation of natural Pisum sativum plants with fruits having an optimum globulin content,specifically with a given vicilin::convicilin ratio naturally modifying the gelation and yieldingpea protein isolates and compositions with a high degree of gelation, without the need ofchemical functionalization and/or cumbersome downstream purification represents astrong need to provide healthy and well standardized pea protein compositions suitable for 5a variety of different food applications.

Further, there is a great need in providing raw materials for alternative and hybrid foodproducts to contribute to a reduced energy consumption during food production incomparison to animal-based food products and to guarantee an alternative source of foodmaterials for a balanced and healthy nutrition. To this end, there is a great need in providing 10methodologies to provide plant-based lines within a short period of time that produce fruits,seeds and materials that can be planted and cultivated in an environmentally friendly wayand which are suitable as valuable basis for alternative nutrition.

Summary of the Invention Brief Description of the Drawings 15 Figure 1 ( Fig. 1 ) shows a general and exemplary flow scheme for producing a pea proteinisolate as further detailed in Example 3 .

Figure 2 ( Fig. 2A to 2E ) visualizes certain steps and products of the workflow detailed in Fig. 1 ( Fig. 2C = protein pellet; Fig. 2D spray drying) until a protein isolate is obtained in Fig. 2E for various starting materials tested. Details are explained in Example 3 . 20 Figure 3 ( Fig. 3 ) provides a very specific workflow for the flow scheme as generallydepicted in Fig. 1 . Details are again explained in Example 3 .

Figure 4 ( Fig. 4A to 4D ) shows the results of a storage protein measurement for the lineKWS 1 as detailed in Example 4 . Notably, Fig. 4A shows the peaks obtained for theproteins of different size (kDa) measured and Fig. 4B shows the corresponding SDS- 25PAGE. Fig. 4C and Fig. 4D show the calculation of proteins obtained. Fig. 4C highlightsvicilin with signal peptide (no. 11, 51.2 kDa) and convicilin with signal peptide (no. 12, 73.2kDa), whereas Fig. 4D additionally marks vicilin without signal peptide (no. 10, 45.4 kDa).For vicilin to convicilin ratio determinations, all available forms as detectable and ashighlighted in Fig. 4D were considered. 30 Figure 5 ( Fig. 5A to 5C ) shows the results of a storage protein measurement for the lineKWS 2 as detailed in Example 4 . In all experiments of Fig. 5A to 5F , vicilin and convicilin was identified both in the form with and without signal peptide. The sum of vicilins (withoutand with signal peptide) and the sum of convicilins (without and with signal peptide),whenever both species could be identified in a pea protein measurement was used todefine vicilin to convicilin ratio as used herein. Figure 5 ( Fig. 5D to 5F ) then shows theresults of a storage protein measurement for the line Proklam as also detailed in Example 5 4 . Also for this measurement, vicilin and convicilin was identified both in the form with andwithout signal peptide. The sum of vicilins (without and with signal peptide) and the sum ofconvicilins (without and with signal peptide), whenever both species could be identified ina pea protein measurement was used to define vicilin to convicilin ratio as used herein (see Fig. 5F ). 10 Figure 6 ( Fig. 6A to 6H ) provides the visualization of the agar ranking test as standardizedgelling test as detailed in Example 5 and corresponding to the measurements as shown in Table 3 and providing a convenient tool to determine and compare the gelation propertiesand pattern of different gels obtained from different pea lines. Top row ( Fig. 6A to 6D )shows the direct measurement for KWS1, KWS2, KWS3, and KWS Proklam, respectively. 15The bottom row ( Fig. 6E to 6H ) shows the follow-up measurement for KWS1, KWS2,KWS3, and KWS Proklam, respectively, after 24 h.

Definitions An "alternative consumable product ingredient" as used herein refers to an ingredient froma plant, including a protein ingredient, which is suitable as part of a "consumable product" 20or of a "consumable composition". Further, at least a part or fraction of the "alternativeconsumable product ingredient" is suitable as a substitute for a commonly known andcommonly fabricated product ingredient, usually it is suitable as an alternative to, forexample, animal-derived or animal-produced ingredients or food, including meat, eggs ordairy. 25 An "alternative food" or "alternative nutrition" or "alternative (food) product / composition"as used herein refers to a food, including liquid food like beverages, which is usually aplant- or microorganism-based food that is an alternative to animal-derived food, includingmeat or dairy. Alternative food products may be of particular interest as an alternativesource of proteins, but the term alternative food refers to any kind of nutritional building 30block, including proteins, carbohydrates, lipids, vitamins, minerals, fibers and the like thatare suitable for food production and that are well accepted or even healthy as food andfeed for human beings or farm animals and pets. An alternative food product or alternativefood thus represents an "alternative consumable product" or an ingredient thereof. The ingredients of the present invention are useful to be implemented into an alternative food,an alternative feed or an alternative cosmetic.

An "alternative food product" as used herein may be present in liquid form, in semi-liquidform or in solid form. As used herein, an "alternative food product" refers to a product forhuman or animal consumption that is usually made with ingredients from animal sources, 5but in which the animal-sourced ingredient has been partially or fully replaced with a plant-based substitute ingredient. Non-limiting examples of alternative food products includealternative beverages such as a milk substitute or a drinkable yogurt substitute, oralternative food products such as an egg, beef, dairy, poultry, or seafood substitute. Otheralternative food products include pet or animal feed products in which some or all of the 10animal-sourced ingredients are substituted with plant-based ingredients. The replacementor substitution of the animal-sourced ingredient may in some embodiments be e.g. morethan 70%, more than 80%, more than 90%, or more than 95%. In other embodiments, thereplacement or substitution of the animal-sourced ingredient may be 70% or less, such as60%, 50%, 40%, etc. Alternative food products may also include non-dairy beverages such 15as sports drinks or smoothies. Alternative food products may further refer to alternativenutritional products, such as plant-based powder, to be used as dietary or nutritionalsupplements.

An alternative product or an alternative composition as used herein, and any pea proteiningredient suitable for the production thereof of the present invention is specifically 20processed (industrially and/or mechanically and/or chemically and/or enzymatically) and apea protein ingredient of this invention will usually be processed, isolated, concentratedand/or otherwise treated for inclusion in an alternative product or composition. Additionallya pea protein ingredient of this invention will usually represent an intermediate ingredient,that was or that can be isolated from a plant representing one part or fraction of a final 25product or composition, or of a mixture or hybrid product. In an alternative product orcomposition, the pea protein ingredient of this invention as alternative part or fraction of thealternative product or composition thus substitutes a part or fraction that would be presentin a commonly known and commonly fabricated product or composition, preferably,wherein it substitutes a part or fraction of animal or non-plant origin in the corresponding 30commonly known and commonly fabricated product.

Whenever the terms "composition" or "pea protein composition" is used herein, it refers toa composition of any of the protein ingredients, flours, concentrates or isolates disclosedherein that are used in an extracted form together with other ingredients from differentorigins to provide said composition. An alternative food or a cosmetic is thus also a 35 composition in this sense. In this context, a "mixture" or "pea protein mixture" is a specificform of a composition, wherein pea protein fractions from different peas or even fromdifferent plants or other sources are mixed with each other to provide a basic proteinmixture (and optionally further additives or ingredients of different nature and/or origin)comprising pea protein of the present invention. 5 A "consumable product" as used herein refers to goods that are usually understood to beused up or depleted during normal business operations, such as food and beverage, officesupplies, cleaning and sanitary products, and medical supplies. In line with the generalunderstanding, there are two main types of consumables: durable consumables, which areexpected to last over a long period of time, and non-durable consumables, which are 10expected to be used up relatively quickly. Examples of consumables include perishablefoods and beverages, paper products, ink cartridges, cleaning chemicals, gloves, andsyringes. The consumable products particularly dealt with herein are non-durableconsumables that are non-toxic when swallowed or applied on the human or animal bodybased on their intended use that are usually made of at least one organic raw materials 15(and optionally others), including food, beverages, gels, ointments, tooth paste and the like.

A "consumable product /composition" according to the present invention represents analternative product or composition comprising at least one alternative plant-basedingredient, preferably a protein ingredient, according to the present invention, preferably apea-based ingredient. 20 As used herein, "dairy substitute" or "dairy substitute composition" or "dairy alternative" or"dairy alternative product" refer to compositions that mimic the general appearance,nutritional content, and/or taste of dairy products produced using animal milk productswithout containing animal-based milk or being substantially free of animal-based products,and includes hybrid products made with lab-grown, fermented and animal-based 25components such as protein components. The dairy substitute may be completely free ofany animal-based milk or animal-based milk protein or almost free of any animal-basedmilk protein, such as e.g. 90% free, or 95% free of any animal-based milk protein. The dairysubstitute may be a dairy-free cheese, a dairy-free yogurt, a dairy-free ice cream, and thelike. 30 Notably, the term "food" as used herein refers to a food intended for human nutrition,whereas a "feed" as used herein refers to a feed.

As used herein, "meat substitute" or "meat substitute composition" or "meat alternative"refers to compositions that mimic the general, organoleptic, and/or nutritional properties ofconsumable products produced using any type of meat or meat analog, including meat,fish, poultry, lab-grown and fermented meat products. This definition includes hybridproducts made with lab-grown, fermented and animal-based components, such as protein 5components. A similar definition is used herein for "egg substitute", "egg substitutecomposition," and "egg alternative".

A "gene" as used herein refers to the coding region of a gene, the non-coding region aswell as upstream and/or downstream located regulatory sequences, including an enhancer,silencer, promoter elements (e.g., proximal, distal and core promoter elements), and 5` 10and/or 3`UTRs. Therefore, a modification of a gene may also include the modification of anon-coding and/ or of a regulatory sequence thereof.

A "hybrid composition / product" or a "hybrid alternative (consumable, includingfood/cosmetic etc.) composition / product" as used herein refers to an alternative productthat at least partially comprises an "alternative food" or "alternative nutrition" or "alternative 15(food) product" comprising a plant-originating protein substance according to the presentinvention, but which may comprise further ingredients.

A "knock-down" of a gene refers to an experimental technique by which the expression ofthe gene (i.e., the transcription from DNA to RNA and thus the amount of active RNAtranscripts) is reduced. A reduced expression can e.g., be achieved by gene silencing 20reducing or abolishing the transcription rate and thus decreasing the amount of functionalRNA available.

A "knock-out", on the other hand, leads to an abolished expression (transcription /translation), i.e., the gene is not fully or wrong transcribed so that expression is abolishedat all or (on protein level) to such an extent that nearly no functional protein is expressed, 25or is expressed very level at or about the detection limit. This can e.g., be achieved byreplacing or interrupting the sequence of the target gene. For example, an early orpremature additional stop codon, preferably close to the start codon can lead to apremature transcription stop that results in the complete loss of functionally translatedprotein. Alternatively, a "knock-out" can be achieved by a mutation leading to a variation in 30the naturally occurring splice donor or splice acceptor site of a eukaryotic gene comprisingexons and introns. The splice donor site usually includes an almost invariant sequence GUat the 5' end of the intron, within a larger, less highly conserved region. The splice acceptorsite at the 3' end of the intron terminates the intron with an almost invariant AG sequence.

If these conserved sequences are mutated, RNA splicing is modified, which may also resultin a knock-out as measured on a transcript (RNA) or translation (protein) level.Consequently, a single targeted nucleotide exchange, deletion or insertion may result in afunctional knock-out in the sense that the sequence encoded by a gene is no longertranscribed or translated. A "knock-out" may also be produced by a deletion of a gene, or 5of a substantial part thereof, on a genomic level, optionally accompanied by a substitutionagainst another sequence.

A "mutation" or a "genome modification" in the context of the present invention refers to anychange of a coherent nucleic acid sequence by modifying a nucleic acid sequence at agiven nucleotide sequence position that results in at least one difference in the (nucleic 10acid) sequence distinguishing it from the original sequence. In particular, a modification canbe achieved by insertion or addition of one or more nucleotide(s), or substitution or deletionof one or more nucleotide(s) of the original sequence or any combination of these.

A "nucleic acid construct", "construct" or "expression construct" refers to a nucleic acidmolecule encoding or comprising one or more genetic elements, which upon introduction 15into a target cell can be transcribed and/or translated into a functional form, e.g., RNA(s)or polypeptide(s) or protein(s). A nucleic acid construct may also comprise regulatorysequences such as promoter and terminator sequences facilitating expression of thegenetic element(s) as well as spacers and introns. The genetic elements of the presentinvention can also be encoded on a set of constructs, which constructs can be introduced 20into a cell simultaneously or consecutively.

A "(pea) protein flour" as used herein refers to an ingredient that contains milled peas.

"Protein texturate" or "textured protein" as used herein refers to an ingredient having astructural integrity and identifiable structure such that individual units, appearing as fibers,shreds, chunks, bits, granules, slices, and the like, will withstand hydration and cooking or 25other procedures used in the production of food for consumption. In general, texturedproteins may be used to alter or enhance texture and bind water. Edible protein sourcesfrom which textured proteins are produced may include, but are not limited to, legumes(e.g., pulse protein), pea, soy, com, wheat, chickpea, potato, rice, sunflower, and the like.Textured proteins may include, but are not limited to, textured pea protein, textured soy 30flour, textured soy concentrate, textured wheat protein, textured potato protein, orcombinations thereof. Methods for protein texturization are known and described in the art,and may include, for example, high temperature and pressure extrusion, spinning, freezetexturization, chemical or enzymatic texturization, and the like.

The term ʺRNAi ʺ or "RNA silencing" or "gene silencing" as used herein interchangeablyrefer to the process called RNA interference meaning a gene down-regulation (or knock-down) mechanism meanwhile demonstrated to exist in all eukaryotes. The mechanism wasoriginally recognized and described in plants where it was called ʺpost-transcriptional genesilencing ʺ or ʺPTGS ʺ. In RNAi, small RNAs function to guide specific effector proteins to a 5target nucleotide sequence by complementary base pairing resulting in degradation of thetarget. A "gene silencing construct" or "RNAi agent" usually comprises so called "sense"and "antisense" sequences. Sense and antisense sequences are complementarysequences, which are present in reverse orientation in a nucleic acid sequence. If a nucleicacid construct comprises a sense and a corresponding antisense sequence, the two 10complementary sequences form an RNA double strand upon transcription, which results inan "RNA hairpin". In an RNA hairpin, sense sequences and corresponding antisensesequences, together form a double strand and are separated by an "intervening intron loopsequence" forming the loop of the hairpin structure.

The terms Pisum sativum (L.) plant and pea plant, and short only pea, are used 15interchangeably herein, wherein the term pea is used in the context of the plant as a whole,but also to denote parts thereof, particularly seeds/fruits within pea pods.

A "pea plant ingredient" as used herein is to be understood as the total amount of proteinthat can be extracted from a pea fruit or seed (dry or fresh). A "(pea) protein ingredient" isin turn to be understood as the total amount of protein of the (pea) plant. 20 The term "protein concentrate" is a protein ingredient with a concentration of about 30% to60%. A "protein isolate" is an even more concentrated protein ingredient with aconcentration of about 60% to about 100%. A "protein flour" represent the protein that canbe obtained directly after dehulling and milling. As this protein flour is not yet heavilyprocessed, it reflects the original content of protein ingredients rather directly. Therefore, 25the "protein flour" was also used to define standard ratios by the inventors when comparingdifferent material herein below. A "protein texturate" or "texturized vegetable protein" isused to describe a usually further defatted flour product that is particularly suitable andused as a meat analogue or meat extender. It is quick to cook, with a protein contentcomparable to some meats. The terms "protein flake" or "protein powder" further describe 30the form of the protein. A "protein powder" is usually composed of fine, dry particlesproduced by the grinding, crushing, or disintegration of a solid substance.

The term "vector" refers to an element used for introducing a nucleic acid construct or setof nucleic acid constructs into a cellular system. The vector may be a plasmid or plasmid vector, cosmid, artificial yeast artificial chromosomes (YAC), bacterial artificialchromosome (BAC) or P1 artificial chromosomes (PACs), phagemid, bacterial phagebased vector, a modified viral vector, an Agrobacterium shuttle vector, an isolated single-stranded or double-stranded nucleic acid sequence, comprising DNA and RNA sequencesin linear or circular form, or a mixture thereof, for introduction or transformation into a plant, 5plant cell, tissue, organ, or material according to the present disclosure.

The terms "plan" or "plant cell" or "part of a plant" as used herein refer to a plant organism,a plant organ, differentiated and undifferentiated plant tissues, plant cells, seeds, andderivatives and progeny thereof. Plant cells include without limitation, for example, cellsfrom seeds, from mature and immature cells or organs, including embryos, meristematic 10tissues, seedlings, callus tissues in different differentiation states, leaves, flowers, roots,shoots, male or female gametophytes, sporophytes, pollen, pollen tubes and microsporesand protoplasts etc.

"Mutagenesis" refers to a technique, by which modifications or mutations are introducedinto a nucleic acid sequence in a random or non- site-specific way. For example, mutations 15can be induced by certain chemicals such as EMS (ethyl methanesulfonate) or ENU (N-ethyl-N-nitrosourea) or physically, e.g., by irradiation with UV or gamma rays. "Site-specificmodifications", on the other hand, rely on the action of site-specific effectors such asnucleases, nickases, recombinases, transposases, base editors, prime editors and the like.These tools recognize a certain target sequence and allow to introduce a modification at a 20specific location within the target sequence.

A "protein composition" as used herein refers to a protein isolate directly obtainable from afruit, seed, particularly from a specific pea of the genus Pisum, or to a flour, a proteinfraction, a purified or partially purified protein fraction. The protein composition, dependingon the way of preparing the same, may include denatured and/or partially fragmented 25proteins, as the proteins may have undergone denaturation and/or fragmentation duringthermal, chemical and/or mechanical processing/purification. A protein composition mayconsist of substantially one protein or a fragment thereof, particularly convicilin, or it maybe a protein composition mixture comprising other proteins, particularly globulins andfurther pea proteins. 30 "TILLING" (Targeting Induced Local Lesions in Genomes) is a process, which allows toidentify mutations in a specific gene after an (unspecific) mutagenesis has been performed.Mutagenesis may e.g., be performed using a chemical mutagen such as EMS. Then, a sensitive DNA screening technique is used to identify single base mutations. Methods forperforming TILLING are known to the skilled person.

A "functional homolog(ue)" as used herein refers to a molecule having substantially thesame function as a reference molecule. A "structural homolog(ue)" refers to a homologhaving a substantial degree of sequence identity to a reference molecule, or a part thereof 5it originates from.

Whenever the present disclosure relates to the percentage of identity of nucleic acid oramino acid sequences to each other, these values define those values as obtained by usingthe EMBOSS Water Pairwise Sequence Alignments (nucleotide) programme(www.ebi.ac.uk/Tools/psa/emboss_water/) nucleic acids or the EM-BOSS Water Pairwise 10Sequence Alignments (protein) programme (www.ebi.ac.uk/Tools/psa/emboss_water/) foramino acid sequences. Alignments or sequence comparisons as used herein refer to analignment over the whole length of two sequences compared to each other. Those toolsprovided by the European Molecular Biology Laboratory (EMBL) European BioinformaticsInstitute (EBI) for local sequence alignments use a modified Smith-Waterman algorithm 15(see www.ebi.ac.uk/Tools/psa/ and Smith, T.F. & Waterman, M.S. "Identification ofcommon molecular subsequence" Journal of Molecular Biology, 1981 147 (1):195-197).When conducting an alignment, the default parameters defined by the EMBL-EBI are used.Those parameters are (i) for amino acid sequences: Matrix = BLOSUM62, gap openpenalty = 10 and gap extend penalty = 0.5 or (ii) for nucleic acid sequences: Matrix = 20DNAfull, gap open penalty = 10 and gap extend penalty = 0.5. The skilled person is wellaware of the fact that, for example, a sequence encoding a protein can be "codon-optimize’’if the respective sequence is to be used in another organism in comparison to the originalorganism a molecule originates from.

Description of the Invention 25 The present invention is directed to improvement of the viscoelastic properties of peaproteins, in particular gelation, and identifying and optimizing the pea plant genetics thatare involved in obtaining desirable viscoelastic performance of pea proteins, namely in theprovision of pea protein ingredients having an increased gelation pattern in comparison tostandard pea lines making the pea protein ingredients highly attractive for alternative food 30and cosmetics production, especially for products and compositions, where a strong textureand an adjustable good gelation is needed. Particularly, the present invention is based onthe finding that specific variants of the protein convicilin and a certain ratio of vicilin toconvicilin can favorably influence the gelation pattern of a pea protein mixture of a pea plant so that alternative foods and consumable products can be made with specific pea linescharacterized by a favorable convicilin content. Further, methods of screening for anproviding suitable pea lines are provided strongly facilitating the provision of pea proteiningredients with a standardized and predictable altered gelation pattern.

In one aspect, there is thus provided a method of altering a gelation pattern of the pea 5protein ingredient of a Pisum sativum plant, the method comprising the following steps: (i)providing at least one pea comprising at least one nucleic acid molecule comprising at leastone modification of a nucleotide sequence of at least one gene, including the regulatoryregion thereof, wherein the at least one gene modified encodes a convicilin protein, whereinsaid at least one modification affects the gelation pattern of the pea protein ingredient, 10wherein said at least one modification affects at least one nucleotide sequence of aPsat06G0558100 reference gene (SEQ ID NO: 2) in comparison to the variantPsat06G0558100 reference gene (SEQ ID NO: 1) and/or of a Psat06G0558200 referencegene (SEQ ID NO: 66) in comparison to the variant Psat06G0558200 reference gene (SEQID NO: 65), or of a combination thereof; (ii) optionally: cultivating the Pisum sativum plant 15to obtain at least one pea comprising a protein composition having an altered gelationpattern in comparison to a Pisum sativum plant not comprising the at least one modifiedconvicilin gene, or the regulatory region thereof; and (iii) obtaining a pea protein ingredientof said Pisum sativum, wherein said pea protein ingredient obtained, preferably asintermediate, has an altered gelation pattern, wherein the altered gelation pattern is defined 20as an altered vicilin to convicilin ratio in a pea protein ingredient, preferably in a pea flour,in comparison to a reference non-processed vicilin to convicilin ratio of 2:1 to 4:1 (wt./wt.)of a pea protein ingredient, preferably a flour, of a reference Pisum sativum plant, whereinthe altered vicilin to convicilin ratio is characterized by a decreased amount of convicilin incomparison to the amount measured for the reference Pisum sativum plant and thus an 25increased vicilin to convicilin ratio. Preferably, the above method is not an essentiallybiological method, as at least one non-naturally occurring SNP, or gene or portion thereofhas to be introduced into a reference gene.

Notably, the present inventors surprisingly found that by modifying the amount of totalconvicilin in a pea and particularly the ratio of vicilin and convicilin resulting from this 30targeted modification has a significant impact on the gelation, i.e., the viscoelasticproperties, of the total protein ingredients obtainable from a pea by defining, providing andtesting the relevant modified variants that show a reduced or even abolished transcriptionand/or translation of the (usually two, or more) genomic copies of convicilin. Altering in thecontext of the present invention thus preferably means a reduction of convicilin and thus 35an increase of gelation properties of the protein ingredients, optionally processed and purified, when used in various compositions, preferably when used in alternative food,where high-value proteins from plants with excellent texture and stability are of greatinterest.

Vicilin, as all pea globulins, comprises a signal peptide for import into the endoplasmicreticulum (ER), which is later on co-translationally removed. Vicilins have two cleavage 5sites, depending on the isoform (A and B. Cleavage yields fragments of about 20 kDa(alpha), 13 kDa (beta) and 12-16 kDa (gamma). Cleavage at site A yields fragments ofabout 20 kDa (alpha) and 25-30 kDa (beta + gamma), whereas cleavage at site B yieldsfragments of about 30-36 kDa (alpha+ beta) and 12-16 kDa (gamma). For convicilins, nopost- or co-translational modifications other than the removal of the signal peptide also 10comprised for this pea globulin was described (source: Tzitzikas et al., 2006, doi:10.1021/jf0519008).

During testing of a plethora of pea flours of various origin, it turned out that – depending onthe stage of harvesting – vicilin and convicilin were usually present in the pea materialtested with and without signal peptide. As the gelation properties of the proteins with and 15without signal peptide are not significantly influenced by the signal peptide, the presentinventors established a robust and easily reproducible test to measure the gelation of pea-derived proteins, usually in a less processed pea flour composition, and identified that theratio of total vicilin (i.e., vicilin with and without signal peptide) to total convicilin (with andwithout signal peptide) represents a reliable indicator to measure the gelation strength and 20thus the gelation pattern of a given pea material having a certain convicilin-expressionsignature. To have a uniform basis of calculation (given the fact that particularly convicilinwas identified in the signal peptide fused form only in certain lines, see Fig. 5 ), it wasdecided that the vicilin to convicilin ratio as used herein thus represents the sum of totalvicilin and/or convicilin respectively, without cleavage products (for vicilin), but always 25caluculating the sum of vicilin and convicilin both with and without the signal peptide (cf. Fig. 4C , Fig. 4D and Fig. 5C : peaks at ~ 45 kDa = vicilin without signal peptide and ~ 51kDa correspond to vicilin with signal peptide the sum of which is used to calculate totalvicilin; whereas for convicilin used in determining the ration corresponds to peaks a about64.7 kDa and 73.2 kDa without and with signal peptide, respectively, Fig. 5C showing both 30convicilin forms as no. 13 and 14, respectively, for KWS line 3, whereas for KWS line 1,only the convicilin with signal peptide was identified as no. 12 (73.2 kDa) in Fig. 4C and Fig. 4D ). As the calculation of total vicilin / convicilin with and without signal peptide (butnot including potential cleavage forms not frequently identified during early processingwhen producing pea flour test samples) is easier to reproduce and showed to be more 35robust, the vicilin to convicilin ratio as disclosed herein is the ratio of vicilin to convicilin measured in total protein or protein flour fractions of pea and determined as detailed in theExamples and as represented for exemplary measurements in the Figures ( Fig. 4 and Fig. 5 ).

The altered gelation pattern as achieved by modifying the convicilin amount in a pea proteiningredient based on the teachings of the present invention thus represents a favorable plant 5trait of interest, as the respective plants are highly suitable for providing proteins useful formaking alternative food or stabilizers in cosmetics having a natural origin.

In one embodiment of the above aspect, there is provided a method, wherein the amountof convicilin in the pea protein ingredient, preferably in the pea flour, is reduced by at least5%, at least 10%, at least 15%, at least 20%, at least 25%, preferably at least 30%, at least 1035%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70%,more preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 100%, even more preferably at least 200%, at least 250%, at least 300%, at least400%, at least 500%, and most preferably almost completely lost in comparison to theamount measured for the reference Pisum sativum plant. 15 According to the various aspects and embodiments of the present invention, there areprovided sequences and methods to obtain a Pisum sativum plant with a convicilin peaprotein encoding sequence that can be modified in a targeted way depending on thegelation pattern needed. The present invention provides a scheme to modify convicilingenes / alleles in a step-by-step way to achieve an adjustable decrease of convicilin 20expression that is correlated with an increased gelation to provide suitable pea proteiningredients with a desired gelation pattern.

The reference Pisum sativum plant as also characterized herein below in direct comparisonto pea lines established and tested according to the present invention is the pea lineProklam that is publicly available and well characterized. 25 All Psat locus information provided herein is based on the nomenclature and annotation aspresented by Yang et al., Nat. Genet., 2022, https://doi.org/10.1038/s41588-022-01172-2).Psat06G0558100 (SEQ ID NO: 1), for example, lies on chromosome 6 of the referencegenome line ZW6 between position 448208288 and 448211033 based on the annotationof said reference genome. 30 The method of claim 1 or 2, further comprising a step of processing, preferably including astep of extraction, and/or purifying the pea protein ingredient of said Pisum sativum planthaving an altered gelation pattern, wherein the processing preferably at least includes a step of dehulling, to obtain a protein flour, a protein concentrate, a protein isolate, a peaprotein texturate, a pea protein powder, or a pea protein flake of a Pisum sativum plant.

In one embodiment, there is provided a method according to the above first aspect, whereinthe convicilin protein has a sequence selected from any one of SEQ ID NOs: 121 and 122,or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 595%, 96%, 97%, 98%, or at least 99% sequence identity thereto, and/or wherein the vicilinprotein preferably has a sequence of SEQ ID NO: 123 or a sequence having at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%sequence identity thereto. As detailed above, and as it is known to the skilled person, othervicilin and convicilin analogoues, homologues, orthologues and paralogues and, 10particularly for vicilin, smaller cleavage products may exist. SEQ ID NO: 123 thusexclusively serves as reference sequence for one representative vicilin protein from Pisumsativum not cleaved into its subunits.

In a first alternative aspect, there is provided a method of improving gelation properties ofa pea protein, comprising the steps of breeding a pea plant line comprising a mutation, 15deletion, or suppression of a convicilin gene, thereby modulating a storage protein contentof peas harvested from the pea plant.

In one embodiment, the mutation, deletion, or suppression of the convicilin gene is of aPsat6g206160 gene, a Psat6g206200 gene, or a combination thereof. The gene can belocated on chromosome 6 or 2 of a pea plant, but homologues, analogues, orthologues 20and paralogues may be located on different chromosomes in different pea plants.

In another embodiment, the Psat6g206160 gene is selected from the group consisting ofa) a nucleic acid molecule of SEQ ID NO: 124, preferably an isolated nucleic acid moleculeand b) a nucleic acid molecule having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 2592%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the nucleicacid molecule of SEQ ID NO: 124 or a functional fragment thereof, and c) a nucleic acidmolecule encoding an amino acid molecule of SEQ ID NO: 125 or a functional fragmentthereof, and d) a nucleic acid molecule encoding an amino acid molecule having at least55%, at least 60%, at least 65%, at least 70%, at least 75%, 76%, 77%, 78%, 79%, 80%, 3081%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or at least 99% sequence identity identity to SEQ ID NO: 125 or afunctional fragment thereof, and e) a nucleic acid molecule hybridizing under stringentconditions to a fragment of at least 250 bases complementary to SEQ ID NO: 124 or a functional fragment thereof, and wherein the Psat6g206200 gene is selected from thegroup consisting of f) the nucleic acid molecule of SEQ ID NO: 126, and g) a nucleic acidmolecule having at least 70%%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or at least 99% sequence identity to the nucleic acid molecule of SEQ ID 5NO: 126 or a functional fragment thereof, and h) a nucleic acid molecule encoding an aminoacid molecule of SEQ ID NO: 127 or a functional fragment thereof, and i) a nucleic acidmolecule encoding an amino acid molecule having at least 55%%, at least 60%, at least65%, at least 70%, at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% 10sequence identity to SEQ ID NO: 127 or a functional fragment thereof, and j) a nucleic acidmolecule hybridizing under stringent conditions to a fragment of at least 250 basescomplementary to SEQ ID NO: 126 or a functional fragment thereof.

In one embodiment, the mutation, deletion, or suppression of the convicilin gene modulatesthe storage protein content of the peas by altering the ratio of convicilin to vicilin proteins 15in the peas to result in improved gelation properties of the pea protein.

In yet a further embodiment, the mutation, deletion, or suppression of the convicilin geneis achieved by mutagenesis, preferably wherein the mutation, deletion, or suppression ofthe convicilin gene is achieved by genetic engineering or TILLING.

In a further alternative aspect, there is provided a method of making a plant protein 20ingredient, comprising selecting the pea plant line obtained by the method of the firstalternative aspect.

In one embodiment of the further alternative aspect, the method further comprises the stepsof cultivating pea plants from the selected pea plant line, harvesting peas from the peaplants, and extracting pea proteins from the harvested peas, preferably wherein the method 25is non an essentially biological method.

In a third alternative aspect, there is provided a method of making a consumable productcomprising the plant protein ingredient produced by the method of the above alternativeaspects.

In a fourth alternative aspect, there is provided a consumable product made by the method 30of the third alternative aspect, wherein the consumable product has improved gelationproperties compared to a consumable product comprising a conventional plant proteiningredient not made by cultivating the selected pea plant line, preferably wherein the consumable product is selected from the group consisting of a food product, a beverageproduct, a nutraceutical product, a pharmaceutical product, a healthcare product, or adietary supplement product, or a combination thereof.

In yet another embodiment, there is provided a method, wherein the at least onemodification is a deletion, substitution, or an insertion, preferably a targeted substitution, of 5at least one nucleotide sequence of the at least one gene, including the regulatory regionthereof, encoding the convicilin protein.

In one embodiment of the methods as described herein, the at least one modification isprovided by a step of mutagenesis, wherein mutagenesis includes chemical mutagenesis,radiation mutagenesis, and genome editing, wherein genome editing includes editing by 10site-directed nucleases, including zinc-finger nuclease (ZFNs) systems, transcriptionactivator-like effector nuclease (TALENs) systems, meganuclease systems andCRISPR/Cas systems, preferably wherein mutagenesis is resulting in a knock-down, morepreferably a knock-out of at least one gene encoding convicilin.

According to the embodiments as disclosed herein, site-directed and random mutagenesis 15are thus encompassed by the term mutagenesis. Mutagenesis may also refer to naturalmutagenesis occurring as natural phenomenon. Based on the screening and selectionmethods as provided herein, such mutagenesis events in a convicilin gene can be easilydetermined to identify interesting germplasm carrying at least one signature mutationaccording to the present invention. 20 According to all aspects of the present disclosure, the Pisum sativum gene encodingconvicilin may be located on any chromosome of a Pisum sativum plant. The relevant generegion can be easily identified. The altered expression of the gene or genes can beachieved by mutagenesis, TILLING, or genetic engineering. Identification and selection ofplants comprising genes with an altered expression can be performed using marker- 25assisted selection using random or functional DNA markers based on the sequence andthe position information provided herein.

In one alternative aspect, there is provided a method of producing a plant of the genusPisum, preferably Pisum sativum, having an improved gelation pattern in view of thedecreased convicilin amount expressed and an increased vicilin to convicilin ratio 30according to the first aspect, the method comprising the (a) introduction of; and/or (b)mutagenesis to confer, preferably chemical- or radiation-induced mutagenesis, and/ortargeted mutagenesis, at least one functional copy of the modified nucleotide sequenceselected from any one of SEQ ID NOs: 3 to 64 or 1, having at least one modification in comparison to the reference gene sequence of SEQ ID NO: 2, and/or at least one functionalcopy of the modified nucleotide sequence is selected from any one of SEQ ID NOs: 67 to120, or 65 having at least one modification in comparison to the reference gene sequenceof SEQ ID NO: 66, or any combination the aforementioned modifications of SEQ ID NOs:to 64 and/or SEQ ID NOs: 67 to 120, or a nucleotide sequence having at least 75%, 76%, 577%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the respectivesequence provided that the respective signature mutation as defined in Table 1 and Table 2 is still present, or a nucleic acid molecule comprising at least one modification of anucleotide sequence of at least one gene, including the regulatory region thereof, the gene 10encoding a convicilin protein, wherein the modified nucleotide sequence comprises at leastone signature mutation as defined in Table 1 and Table 2 in comparison to the referencegene sequence of SEQ ID NO: 2 or SEQ ID NO: 66, respectively, into a Pisum plant,preferably a Pisum sativum plant of interest.

As detailed in the Examples, a deep and thorough screen for single nucleotide 15polymorphisms (SNPs) in the convicilin gene (all alleles; various reference genomesscreened) was performed to identify suitable modifications. These were screened for,optionally inserted in a targeted way into test lines, or lines carrying suitable germplasm asidentified after screening were tested. A clear correlation of the SNP mutations and thedesired high gelation pattern due to a decrease in the amount of convicilin proteon 20transcribed or expressed was surprisingly confirmed. Suitable SNPs, reference positionsand the corresponding SEQ ID NOs are shown in Tables 1 and 2 for Psat06G0558100( Table 1 ) and Psat06G0558200 ( Table 2 ) respectively. The SNP position is indicated asexact position in the reference genome.

Table 1 SEQID NO:Position KE_ZW6_hq-v1.chr6Psat06G0558100Reference Modification Effect (genomic level)Effect(proteinlevel)448208301 A G start_lost +++448208303 A G stop_gained +++448208304 A G stop_gained +++448208410 T C stop_gained +++448208437 T C stop_gained +++448208539 T C stop_gained +++448208557 T C stop_gained +++448208594 A G stop_gained +++448208595 A G stop_gained +++448208635 T C stop_gained +++448208653 T C stop_gained +++448208690 A G stop_gained +++448208691 A G stop_gained +++448208710 T C stop_gained +++448208731 T C stop_gained +++448208746 T C stop_gained +++448208776 T C stop_gained +++448208785 T C stop_gained +++448208791 T C stop_gained +++448208809 T C stop_gained +++448208821 T C stop_gained +++448208840 A G stop_gained +++448208841 A G stop_gained +++448208863 T C stop_gained +++448208879 A G stop_gained +++448208880 A G stop_gained +++448208890 T C stop_gained +++448208965 T C stop_gained +++448209004 T C stop_gained +++448209010 T C stop_gained +++448209088 T C stop_gained +++448209127 T C stop_gained +++448209181 T C stop_gained +++448209221 A G splice_donor_variant +++448209372 A Gsplice_acceptor_variant +++448209480 T C stop_gained +++448209549 A G splice_donor_variant +++448209667 A Gsplice_acceptor_variant ++448209695 T C stop_gained ++448209704 T C stop_gained ++448209743 A G splice_donor_variant ++448209816 A Gsplice_acceptor_variant +448209856 T C stop_gained +448209880 T C stop_gained +448209883 T C stop_gained +448209925 T C stop_gained +448210075 T C stop_gained +448210081 T C stop_gained +448210123 A G splice_donor_variant + 52 448210210 A Gsplice_acceptor_variant +448210310 T C stop_gained +448210313 T C stop_gained +448210352 T C stop_gained +448210494 A G splice_donor_variant +448210590 A Gsplice_acceptor_variant +448210614 T C stop_gained +448210659 T C stop_gained +448210686 T C stop_gained +448210692 T C stop_gained +448210722 T C stop_gained +448210731 T C stop_gained +448210740 T C stop_gained + Table 2 SEQID NO:Position KE_ZW6_hq-v1.chr6Psat06G0558200Reference Modification Effect (genomic level)Effect(proteinlevel)448252681 A G stop_gained +448252699 A G stop_gained +448252729 A G stop_gained +448252735 A G stop_gained +448252762 A G stop_gained +448252807 A G stop_gained +448252831 T Csplice_acceptor_variant +448252905 T C splice_donor_variant +448252924 A G stop_gained +448253047 A G stop_gained +448253086 A G stop_gained +448253089 A G stop_gained +448253189 T Csplice_acceptor_variant +448253276 T C splice_donor_variant +448253318 A G stop_gained +448253324 A G stop_gained +448253474 A G stop_gained +448253516 A G stop_gained +448253519 A G stop_gained +448253543 A G stop_gained +448253546 A G stop_gained +448253561 A G stop_gained +448253601 T Csplice_acceptor_variant ++448253702 T C splice_donor_variant ++448253741 A G stop_gained ++448253750 A G stop_gained ++448253778 T Csplice_acceptor_variant ++448253880 T C splice_donor_variant ++448253949 A G stop_gained +++448254057 T Csplice_acceptor_variant +++448254207 T C splice_donor_variant +++ 98 448254247 A G stop_gained +++448254301 A G stop_gained +++100 448254340 A G stop_gained +++101 448254418 A G stop_gained +++102 448254424 A G stop_gained +++103 448254463 A G stop_gained +++104 448254538 A G stop_gained +++105 448254548 T C stop_gained +++106 448254549 T C stop_gained +++107 448254565 A G stop_gained +++108 448254587 T C stop_gained +++109 448254588 T C stop_gained +++110 448254607 A G stop_gained +++111 448254619 A G stop_gained +++112 448254637 A G stop_gained +++113 448254643 A G stop_gained +++114 448254652 A G stop_gained +++115 448254692 T C stop_gained +++116 448254693 T C stop_gained +++117 448254730 A G stop_gained +++118 448254748 A G stop_gained +++119 448254847 A G stop_gained +++120 448254866 T C start_lost +++ As evident from the + to +++ ranking in the column Effect (protein level) in Tables 1 and 2,a ranking was performed showing the influence of a single signature mutation on level ofexpressed convicilin protein. An early stop codon causes a loss of transcription of RNA andthus a loss of translation to protein and thus, in sum, no transcription/translation at all. This 5leads to a reduced expression of convicilin and consequently to an increased gelling effect.The lower the expression the better is the effect on protein level. +++ gelation reduced by 80 to 100% ++ gelation reduced by 40 to < 80% + gelation reduced by 0 to < 40% 10 In certain embodiments, at least one signature mutation of Table 1 and of Table 2 will becombined. In certain embodiments, more than one mutation will be combined. Inembodiments, where a high gelation is of interest, at least one signature mutation in eachconvicilin encoding gene / allele will be present, preferably each at least a ++, morepreferably each a +++ mutation as detailed above. 15 The total pea protein ingredients produced by these selected pea lines or varieties have alower convicilin content, or even no convicilin, and therefore significantly improved gelation,as compared to pea protein ingredients from plants in which the expression of one or moreof the convicilin biosynthetic pathway genes is not reduced, significantly reduced, or eliminated. The selected pea plants are cultivated, and the peas may be harvested fromthese plants. The protein fraction can be extracted from the harvested peas, resulting in apea protein ingredient having improved gelation properties as compared to a conventionalpea protein ingredient made from peas in which the expression of one or more of theconvicilin genes is not altered. 5 The method of the first aspect can, for instance, be used as part of or in combination withbreeding programs, genome editing, mutagenesis, such as TILLING (Targeting InducedLocal Lesions in Genomes) approaches, or other means of developing of new plant lines,including donor lines, comprising the modified convicilin alleles according to the presentinvention. 10 An allele can be detected by any means known in the art, including hybridization to allele-specific oligonucleotides, such as competitive allele specific PCR (KASP), or simply bynucleotide sequencing of the respective polymorphic region, including next generationsequencing and/or high throughput sequencing methods. Markers and suitable probes,including fluorescent probes, can be easily designed based on the mutation of the SNPs in 15the convicilin gene of particular interest as provided with the present invention.

In certain embodiments, the method may comprise an amplification step, such as PCRamplification or LCR (ligase chain reaction, a technique well known to the skilled person)amplification, to genotype one or more polymorphic positions and/one to detect one or morealleles associated with an improved trait DNA amplifications methods are well known in the 20art.

Embodiments relating to allele-specific amplification, including KASP, may comprise a non-allele-specific amplification step followed by allele-specific amplification or may use allele-specific amplification direction on the provided genomic DNA.

The method according to the present invention may be utilized for marker-assisted 25selection (MAS; Sun et al., 2020), including genomic selection (GS) also referred to asgenome wide selection (GWS).

Chemical- or radiation-induced mutagenesis is well established in the art and may beperformed by any known suitable method, such as but not limited to EMS (ethylmethanesulfonate) mutagenesis or UV mutagenesis. As chemical- or radiation-induced 30mutagenesis is a random mutagenesis approach, expression of convicilin may bemeasured, as disclosed herein or by any suitable method known in the art. Throughmeasuring the expression, mutated one or more plants having an increased expression of convicilin can be identified and/or selected after chemical- or radiation-inducedmutagenesis.

Introduction may be through stable or transient introduction by means of transformationand/or insertion using genome-modification technology. Preferably, the introduction of theat least one functional copy comprised its integrated into the chromosomal plant genome 5and/or the chloroplast genome. Transformation may be performed by any method knownin the art, such as but not limited to agrobacterium-mediated transformation, particlebombardment, PEG-mediated transformation, particle uptake or electroporation.Introduction through genome-modification technology may be performed by any site-specific genome modification technique known in the art using an SDN-3 approach, 10wherein the introduced sequence may be inserted by replacing at least on endogenouscopy of a convicilin encoding gene or may be introduced elsewhere in the genome.

Embodiments using a) introduction and/or b) mutagenesis by genome modification, rely ona genome modification system, wherein a genome modification system refers to any DNA,RNA and/or amino acid sequence introduced into the cell, on a suitable vector and/or 15coated on a particles and/or directly introduced, wherein the genome modification systemcauses the modification of the genome of the cell in which it has been introduced, whereinthe genome modification system comprises at least one site-directed nuclease, nickase orinactivated variant thereof, and optionally at least one further molecule, such as a guidemolecule. 20 A ʺsite-directed nuclease ʺ herein refers to a nuclease or an active fragment thereof, whichis capable of specifically recognizing and cleaving DNA at a certain location, the targetsequence. Such nucleases typically produce a double-strand break (DSB), which is thenrepaired by non-homologous end-joining (NHEJ) or homologous recombination (HR). Site-specific nucleases include meganucleases, homing endonucleases, zinc finger nucleases, 25transcription activator-like nucleases and CRISPR nucleases, or variants includingnickases or nuclease-dead variants thereof.

A "CRISPR nuclease", as used herein, is a specific form of a site-directed nuclease andrefers to any nucleic acid guided nuclease which has been identified in a naturally occurringCRISPR system, which has subsequently been isolated from its natural context, and which 30preferably has been modified or combined into a recombinant construct of interest to besuitable as tool for targeted genome engineering. Any CRISPR nuclease can be used andoptionally reprogrammed or additionally mutated to be suitable for the variousembodiments according to the present invention as long as the original wild-type CRISPR nuclease provides for DNA recognition, i.e., binding properties. CRISPR nucleases alsocomprise mutants or catalytically active fragments or fusions of a naturally occurringCRISPR effector sequences, or the respective sequences encoding the same. A CRISPRnuclease may in particular also refer to a CRISPR nickase or even a nuclease-dead variantof a CRISPR polypeptide having endonucleolytic function in its natural environment. A 5variety of different CRISPR nucleases/systems and variants thereof are meanwhile knownto the skilled person and include, inter alia, CRISPR/Cas systems, including CRISPR/Cas9systems (EP2771468), CRISPR/Cpf1 systems (EP3009511B1), CRISPR/C2C2 systems,CRISPR/CasX systems, CRISPR/CasY systems, CRISPR/Cmr systems, CRISPR/MADsystems, including, for example, CRISPR/MAD7 systems (WO2018236548A1) and 10CRISPR/MAD2 systems, CRISPR/Cas  systems (Pausch et al., Science, 2020,10.1126/science.abb1400), CRISPR/CasZ systems and/or any combination, variant, or 30catalytically active fragment thereof. A nuclease may be a DNAse and/or an RNAse, inparticular taking into consideration that certain CRISPR effector nucleases have RNAcleavage activity alone, or in addition to the DNA cleavage activity. 15 The ʺguide molecule ʺ or ʺguide nucleic acid sequence ʺ (usually called and abbreviated asguide RNA, crRNA, crRNA+tracrRNA, gRNA, sgRNA, depending on the correspondingCRISPR system representing a prototypic nucleic acid-guided site-directed nucleasesystem), which recognizes a target sequence to be cut by the nuclease. The at least oneʺguide nucleic acid sequence ʺ or ʺguide molecule ʺ comprises a ʺscaffold region ʺ and a 20ʺtarget region ʺ. The ʺscaffold region ʺ is a sequence, to which the nucleic acid guidednuclease binds to form a targetable nuclease complex. The scaffold region may comprisedirect repeats, which are recognized and processed by the nucleic acid guided nucleaseto provide mature crRNA. A pegRNAs may comprise a further region within the guidemolecule, the so-called ʺprimer-binding site ʺ. The ʺtarget region ʺ defines the 25complementarity to the target site, which is intended to be cleaved. A crRNA as used hereinmay thus be used interchangeably herein with the term guide RNA in case it unifies theeffects of meanwhile well-established CRISPR nuclease guide RNA functionalities. CertainCRISPR nucleases, e.g., Cas9, may be used by providing two individual guide nucleic acidsequences in the form of a tracrRNA and a crRNA, which may be provided separately, or 30linked via covalent or non-covalent bonds/interactions. The guide RNA may also be apegRNA of a Prime Editing system. The at least one guide molecule may be provided inthe form of one coherent molecule, or the sequence encoding the same, or in the form oftwo individual molecules, e.g., crRNA and tracr RNA, or the sequences encoding the same.

In certain embodiments, a donor plant or donor plant population, comprising one or more 35alleles associated with an improved trait according to the present invention, may be crossed with a recipient plant or a recipient plant population, such as a plant of an elite line or anyplant of interest, to introduce one or more alleles associated with an improved trait in therecipient plant or plant population, e.g. as part of a breeding program. The methodaccording to the present invention may be used to identify one or more progenies of suchcrossings, having an improved trait. 5 In certain embodiments, one or more selected plants are used for one or more furtherbreeding/crossing steps, including backcrossing, the one or more progenies of said one ormore further crossing steps may be selected again for an improved trait by methodsaccording to the invention. Cycles of crossing/breeding and selection of plants having animproved trait according to the present invention may be repeated multiple times. 10 The term "crossed" or "cross" refers to a sexual cross and involves the fusion of two haploidgametes via pollination to produce diploid progeny (e.g., cells, seeds or plants). The termencompasses both the pollination of one plant by another and selfing (or self-pollination,e.g., when the pollen and ovule are from the same plant).

"Backcrossing" refers to the process by which progeny are repeatedly crossed back to one 15of the parents, such as the (donor) parent comprising one or more alleles associated withan improved trait according to the present invention. In a backcrossing scheme, the "donor"parent refers to the parental plant with the desired gene/genes, locus/loci, or specificphenotype to be introgressed. The "recipient" parent (used one or more times) or"recurrent" parent (used two or more times) refers to the parental plant into which the gene 20or locus is being introgressed.

In certain embodiments, one or more plants having at least one improved trait according tothe present disclosure, particularly an altered gelation pattern due to an decreased amountof convicilin expressed, may be identified directly, i.e. one or more parts of the plant,comprising genomic DNA of said one or more plants, is removed and used for the method 25according to the present invention, thereby identifying the presence of one or more allelesassociated with an improved trait in, or representing the situation in, said one or more plantsitself.

In certain embodiments, the method may comprise regeneration of an entire plant,preferably a fertile plant, from a plant cell, preferably derived from somatic tissue, 30embryonic tissue, callus tissue or protoplast. Regeneration may also be somaticembryogenesis, which is an artificial process in which a plant or embryo is derived from asingle somatic cell or group of somatic cells. Somatic embryos are formed from plant cells that are not normally involved in the development of embryos, i.e. plant tissue like buds,leaves, shoots etc.

In certain embodiments, the method comprises the step of analyzing and/or verifying thedesired trait phenotype of one or more selected plants as disclosed herein and/or by usingany suitable method known in the art. 5 In a second aspect, there is provided a nucleic acid molecule comprising at least onemodification of a nucleotide sequence of at least one gene, including the regulatory regionthereof, the gene encoding a convicilin protein, wherein the modified nucleotide sequenceis selected from any one of SEQ ID NOs: 3 to 64 or 1, having at least one modification incomparison to the reference gene sequence of SEQ ID NO: 2, and/or wherein the modified 10nucleotide sequence is selected from any one of SEQ ID NOs: 67 to 120, or 65 having atleast one modification in comparison to the reference gene sequence of SEQ ID NO: 66,or any combination the aforementioned modifications of SEQ ID NOs: 3 to 64 and/or SEQID NOs: 67 to 120, or a nucleotide sequence having at least 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 1595%, 96%, 97%, 98%, or at least 99% sequence identity to the respective sequenceprovided that the respective signature mutation as defined in Table 1 and Table 2 is stillpresent, or a nucleic acid molecule comprising at least one modification of a nucleotidesequence of at least one gene, including the regulatory region thereof, the gene encodinga convicilin protein, wherein the modified nucleotide sequence comprises at least one 20signature mutation as defined in Table 1 and Table 2 in comparison to the reference genesequence of SEQ ID NO: 2 or SEQ ID NO: 66, respectively.

In certain embodiments, the nucleic acid molecule is an isolated nucleic acid molecule.

In certain embodiments, there will be at least one signature mutation as defined in Table 1and Table 2 in comparison to the reference gene sequence of SEQ ID NO: 2 or SEQ ID 25NO: 66, respectively in at least one convicilin gene of a Pisum plant. In anotherembodiments, there will be more than one signature mutation as defined in Table 1 andTable 2 in comparison to the reference gene sequence of SEQ ID NO: 2 or SEQ ID NO:66, respectively in more than one convicilin allele of a Pisum plant, or even in all alleles. Inyet another embodiment, more than one signature mutation as defined in Table 1 and Table 30in comparison to the reference gene sequence of SEQ ID NO: 2 or SEQ ID NO: 66,respectively will be present in one and the same convicilin gene/allele of a Pisum plant.

In yet a third aspect, there is provided an expression construct or a vector comprising atleast one modified nucleotide sequence of at least one convicilin gene and/or of the regulatory regions thereof of the second aspect. Embodiments of this third aspect areparticularly suitable to introduce at least one SNP signature mutation from a plasmid ofinterest into a target cell for studying the effect thereof in vitro and in vivo, preferably byintroducing the modification altering the gelation pattern of a pea protein ingredient in aninheritable way. In another embodiment, transient RNAi constructs may be constructed 5from the expression constructs or vectors to transiently modify the convicilin transcriptionin a cell or plant of interest.

In one embodiment of the third aspect, the present invention relates to an expressionconstruct or vector, or a combination thereof, encoding a genome editing system, whichtargets at least one convicilin gene of a plant of the genus Pisum, or a functional or 10structural homologue thereof, wherein the genome editing system comprises (a) at leastone site-specific nuclease or site-specific nickase, and optionally, in case a CRISPRsystem is used, at least one guide molecule or a sequence encoding the same, and (b)optionally, at least one repair template, or a sequence encoding the same.

In one embodiment of the expression construct or vector described above, the at least one 15genome editing system is selected from a CRISPR/Cas system, preferably from aCRISPR/MAD7 system, a CRISPR/Cpf1 (CRISPR/Cas12a) system, a CRISPR/MAD2system, a CRISPR/Cas9 system, a CRISPR/CasX system, a CRISPR/Cas  system, aCRISPR/CasY system, a CRISPR/Cas13 system, or a CRISPR/Csm system, or the at leastone genome editing system is selected from a zinc finger nuclease system, or a 20transcription activator-like nuclease system, or a meganuclease system, or anycombination, variant, or an active fragment thereof.

In an alternative aspect, the convicilin expression of one or more convicilin genes in aPisum plant can be transiently reduced by a targeted knock-down of the transcription of theone or more convicilin genes to obtain a pea protein plant ingredient of the present 25invention without a modification of the plant on a genomic level. In one embodiment. In oneembodiment of this knock-down method, an RNA silencing construct can be designed,introduced and used to reduce convicilin transcription, wherein said construct is, or theconstruct encodes an RNAi construct comprising a sense and an antisense sequencetargeting a convicilin gene, or a functional or structural homolog thereof, the RNAi construct 30forming an RNA hairpin upon transcription. Suitable constructs for use in planttransformation (stable and transient) are known to the skilled person. Sense and antisenseregion(s) can be designed based on the sequence information provided herein.

The present inventors surprisingly observed that modifying the ratio of convicilin to vicilinstorage proteins results in improved gelation properties, making the pea protein ingredientsuitable for use in many applications. Such applications may include, but are not limited to,a food product, a beverage product precursor (e.g., highly viscous syrup), a nutraceuticalproduct, a pharmaceutical product, a healthcare product, or a dietary supplement product. 5 In a fourth aspect, there is this provided a protein flour, a protein concentrate, a proteinisolate, a pea protein texturate, a pea protein powder, or a pea protein flake of a Pisumsativum plant, having an altered gelation pattern, wherein the Pisum sativum plantcomprises at least one modification of a nucleotide molecule in a nucleotide sequence ofat least one convicilin gene, including the regulatory sequence thereof, wherein the 10nucleotide molecule is as defined in the second aspect above, and/or wherein the Pisumsativum plant, or a cell thereof, comprises at least one expression construct or at least onevector the third aspect above, or wherein the Pisum sativum plant comprises at least onemodified convicilin protein as defined in the above first aspect, wherein the modification istat least one signature mutation. 15 In a fifth aspect, there is provided a pea protein mixture or a pea protein compositioncomprising a protein flour, a protein concentrate, a protein isolate, a pea protein texturate,a pea protein powder, or a pea protein flake of a Pisum sativum plant of the forth aspectand additionally comprising at least one further additive and/or ingredient.

In certain embodiments, the pea protein ingredient, flour, protein concentrate, protein 20isolate, pea protein texturate, pea protein powder, or the pea protein flake of a Pisumsativum plant, having an altered gelation pattern, or any composition or mixture thereof,may be used as an alternative to animal-sourced proteins, resulting in a vegetarian orvegan option for consumers seeking meat or dairy alternatives. Examples of meat-alternative applications include burgers, sausages, and filets made with pea protein to 25replace some or all of the meat ingredients in such products. Examples of dairy-alternativeapplications include cheese-, yoghurt-, and milk-alternatives made with pea protein toreplace some or all of the dairy ingredients in such products. Preferably, the pea proteincomposition having a high gelation pattern as disclosed herein will be particularly suitablefor solid or semi-solid alternative food, or as basis for cosmetics (e.g., gels, crèmes and 30ointments and the like, usually having a semi-solid consistency to be easily applied), as theviscoelastic properties give structure, texture and stability to the product, which is otherwisehard to achieve with protein materials from natural sources.

The pea protein ingredient, flour, protein concentrate, protein isolate, pea protein texturate,pea protein powder, or the pea protein flake of a Pisum sativum plant, having an alteredgelation pattern, produced by the selected pea plants preferably exhibits desiredviscoelastic and other functional properties, particularly gelation, required for suitableextraction, processing, and production for use in consumable applications. In one 5embodiment, the desired properties are directed to the ability of the protein fraction toaggregate, i.e., form cross-linkages to form a stable gel mass that can be, for example, cut,sliced, shredded, or spread in a manner comparable to meat products or, preferably solidand semi-solid, dairy products. The meat products may include but are not limited toground meat analogues, shredded meat analogues, and cut or fileted meat analogues. The 10dairy products may be hard, semi-hard, semi-soft, or soft cheese products, or spoonableor pourable dairy products, also including ice cream and the like.

In certain embodiments, a consumable product, preferably an alternative food product oran alternative cosmetic product, or a hybrid product, as described herein may include oneor more lipid composition(s), for example a fat, an oil, or combinations thereof. In general, 15fats refer to lipid compositions that are solid at room temperature, whereas oils are liquidat room temperature. The lipid compositions may include saturated fatty acids (alsoreferred to as "saturated fats"), unsaturated fatty acids (also referred to as "unsaturatedfats"), or combinations thereof. The lipid composition may include, but are not limited to,vegetable oil, coconut oil, palm oil, sunflower oil, soy oil, canola oil, or combinations thereof. 20The consumable product may include between 1% and 80%, between 1% and 70%,between 1% and 10%, between 1% and 5%, between 5% and 30%, between 10% and25%, between 10% and 75%, or between 15% and 70% by weight of a lipid compositiondepending on the type of dairy substitute. An ordinarily skilled artisan will understand theappropriate lipid composition inclusion rate for a given composition. 25 In certain embodiments, a cosmetic product or composition may additionally includeapproved water-soluble and/or oil-soluble UV-A, UV-B and / or Broadband filter substancesand optionally further additives or ingredients used in cosmetics, for example perfume,dyes, antimicrobials, lipid-replenishing agents, complexing and sequestering agents,pearlescent agents, other plant extracts, vitamins, active ingredients, preservatives, 30bactericides, repellents, self-tanners, depigmenting agents, pigments that have a coloringeffect, softening, moisturizing and / or moisturizing substances, or other usual componentsof a cosmetic or dermatological formulation such as emulsifiers, polymers, foam stabilizersand electrolytes. Preferably, the protein flour, a protein concentrate, a protein isolate, a peaprotein texturate, a pea protein powder, or a pea protein flake of a Pisum sativum plant, 35having an altered gelation pattern is used as a natural stabilizer alternative.

In certain embodiments, the pea protein ingredient, flour, protein concentrate, proteinisolate, pea protein texturate, pea protein powder, or the pea protein flake of a Pisumsativum plant, having an altered gelation pattern, or any composition or mixture thereof,may thus be favorably used as cosmetic thickener or stabilizer, as the natural pea-derivedproduct with increased gelation helps to increase viscosity in cosmetic water and water-in- 5oil based emulsions or to favorably influence the rheology of a cosmetic product orcomposition.

The alternative food product, or the alternative cosmetic product, or a hybrid product, mayinclude water as needed. For example, the product may include between 1% and 80%,between 5% and 75%, between 15% and 70%, between 45% and 65%, between 50% and 1060%, between 1% and 20%, or between 5% and 15% by weight of water depending on thetype of consumable product.

In some embodiments, the consumable product comprising a pea protein ingredient, flour,protein concentrate, protein isolate, pea protein texturate, pea protein powder, or a peaprotein flake of a Pisum sativum plant, having an altered gelation pattern includes both 15water and a plant-based ingredient. The total of the water and plant-based ingredient maybe e.g. between 50% and 95% or between 60% and 90% by weight of the composition.

In certain embodiments, the consumable product comprising a pea protein ingredient, flour,protein concentrate, protein isolate, pea protein texturate, pea protein powder, or a peaprotein flake of a Pisum sativum plant, having an altered gelation pattern may include fiber. 20The fiber may include, but is not limited to, pectin, apple fiber, psyllium, flax fiber, rice branextract, Konjac flour, and the like. The consumable product may include between 0.01%(wt) and 3% (wt), between 0.05% (wt) and 2% (wt), or between 0.1% (wt) and 2% (wt) offiber. The consumable product may include fiber in an amount up to 0.5% (wt), up to 1%(wt), up to 1.5% (wt), up to 2% (wt), up to 2.5% (wt), or up to 3% (wt). 25 In certain embodiments, the consumable product comprising a pea protein ingredient, flour,protein concentrate, protein isolate, pea protein texturate, pea protein powder, or a peaprotein flake of a Pisum sativum plant, having an altered gelation pattern may includestarch. The starch may include a pregelatinized starch, a modified starch, or combinationsthereof. The starch may include, but is not limited to, com starch, potato starch, tapioca 30starch, and the like. The consumable product may include between 0.5% (wt) and 25%(wt), between 1.0% (wt) and 20% (wt), or between 2% (wt) and 18% (wt) of starch. Theconsumable product may include a hydrocolloid. For example, the consumable productmay include guar gum, xanthan gum, locust bean gum, carrageenan, cellulose, konjac gum, and combinations thereof. The consumable product may include between 0.01% and5%, between 0.05% and 4.5%, between 0.1% and 4.0%, or between 0.5% and 3.8% byweight of hydrocolloid. The consumable product may include up to 5%, up to 4.5%, up to4.0%, up to 3.8%, up to 3.5%, up to 2.5%, up to 2.0%, or up to 1.0% by weight ofhydrocolloid. 5 In some embodiments, the consumable product comprising a pea protein ingredient, flour,protein concentrate, protein isolate, pea protein texturate, pea protein powder, or a peaprotein flake of a Pisum sativum plant, having an altered gelation pattern may includelecithin. The consumable product may include between 0.01% and 10%, between 0.05%and 8.0%, or between 0.1% and 5% by weight lecithin. 10 In certain embodiments, the consumable product comprising a pea protein ingredient, flour,protein concentrate, protein isolate, pea protein texturate, pea protein powder, or a peaprotein flake of a Pisum sativum plant, having an altered gelation pattern may include apreservative. For example, the consumable product may include a preservative such as,but not limited to potassium sorbate. The consumable product may include a preservative 15in an amount up to 0.1%, up to 0.5%, or up to 1.0% by weight of the consumable product.The consumable product may include a flavour or seasoning. For example, the consumableproduct may include a natural or artificial flavour(s) and/or seasonings. Seasonings mayinclude, but are not limited to, sweetener(s), salt (e.g., sodium chloride, potassium chloride,and the like), cocoa, chocolate, cinnamon, nutmeg, coconut, almond, combinations thereof, 20and the like. The consumable product may include between 1% and 20%, between 1.5%and 10%, between 5% and 20%, or between 2% and 18% of a flavour or seasoning. Theconsumable product may be free of any flavours or seasoning. In some embodiments, theconsumable product may include between 0.001% and 3.0%, between 0.01% and 2.0%,or between 0.025% and 1.75% of a salt. The consumable product may be free of salt. 25 In yet another embodiment, the consumable product comprising a pea protein ingredient,flour, protein concentrate, protein isolate, pea protein texturate, pea protein powder, or apea protein flake of a Pisum sativum plant, having an altered gelation pattern may includea sweetener. Suitable sweeteners are known and described in the art. The sweetener canbe at least one of a non-caloric sweetener or a caloric sweetener. The sweetener can be 30any type of sweetener, for example, a sweetener obtained from a plant or plant product, ora physically or chemically modified sweetener obtained from a plant, or a syntheticsweetener. Exemplary sweeteners include steviol glycosides, mogrosides, sucrose,fructose, glucose, erythritol, maltitol, lactitol, sorbitol, mannitol, xylitol, tagatose, trehalose,galactose, rhamnose, cyclodextrin (e.g., a-cyclodextrin, b-cyclodextrin, and g-cyclodextrin), 35 ribulose, threose, arabinose, xylose, lyxose, allose, altrose, mannose, idose, lactose,maltose, invert sugar, isotrehalose, neotrehalose, palatinose or isomaltulose, erythrose,deoxyribose, gulose, idose, talose, erythrulose, xylulose, psicose, turanose, cellobiose,glucosamine, mannosamine, fucose, fuculose, glucuronic acid, gluconic acid, glucono-lactone, abequose, galactosamine, xylo-obgosaccharides (xylotriose, xylobiose and the 5like), gentio- obgoscaccharides (gentiobiose, gentiotriose, gentiotetraose and the like),galacto- obgosaccharides, sorbose, ketotriose (dehydroxyacetone), aldotriose(glyceraldehyde), nigero- obgosaccharides, fructoobgosaccharides (kestose, nystose andthe like), maltotetraose, maltotriol, tetrasaccharides, mannan-oligosaccharides, malto-oligosaccharides (maltotriose, maltotetraose, maltopentaose, maltohexaose, 10maltoheptaose and the like), dextrins, lactulose, melibiose, raffmose, rhamnose, ribose,sucralose, acesulfame K, aspartame, saccharin, coupling sugars, soybeanoligosaccharides, and combinations thereof. D- or L-configurations can be used whenapplicable.

The consumable product comprising a pea protein ingredient, flour, protein concentrate, 15protein isolate, pea protein texturate, pea protein powder, or a pea protein flake of a Pisumsativum plant, having an altered gelation pattern may include an acid. Suitable acidsinclude, but are not limited to, citric acid, lactic acid, sorbic acid, malic acid, combinationsthereof, and the like. The consumable product may include an acid in an amount up to0.001%, up to 0.005%, up to 0.01%, up to 0.1%, up to 1.0%, up to 1.5%, or up to 2.0% of 20the consumable product. The consumable product may include between 0.0001% and2.0%, between .0002% and 1.5%, between 0.0003% and 1.0% by weight of an acid.

According to certain embodiments disclosed herein, the alternative product is a hybridproduct that comprises a pea protein ingredient of the present invention as well as at leastfurther additive and/or ingredient. Besides additives and ingredients typically used for food 25and cosmetic production, also at least one lab-grown material can be used. A lab-grownmaterial as used herein refers to a cultured and/or fermented cell, and the material (anybiomass, cellular and non-cellular, including supernantant) obtained therefrom, includingbacterial cells, fungal cells, including filamentous fungi, particularly fungi and fungi biomasssuitable as meat substitute, and and cultured animal cells, including hepatocytes, 30myoblasts, osteoblasts, fibroblasts, lipoblasts, odontoblasts, adult neuronal progenitorcells, neural stem cells, multipotent stem cells from subventricular forebrain region,ependymal-derived neural stem cells, hematopoietic stem cells, liver-derivedhematopoietic stem, marrow-derived stem cell, adipo-fibroblasts, adipose-derived stemcells, islet- cells producing stem cells, pancreatic-derived pluripotent islet-producing stem 35cells, mesenchymal stem cells, placenta cells, bone marrow stromal cells, muscle side population cells, bone marrow-derived recycling cells, blood-derived mesenchymalprecursor cells, bone- marrow derived side population cells, muscle precursor cells,circulating skeleton stem cells, neural progenitor cells, multipotent adult progenitor cells,mesodermal progenitor cells, spinal cord progenitor cells and spore-like cell, and anycombinations thereof, wherein the cell is not derived from a human embryo. 5 In certain embodiments, a hybrid food or composition may thus comprise at least oneportion being a pea protein ingredient, flour, protein concentrate, protein isolate, pea proteintexturate, pea protein powder, or a pea protein flake of a Pisum sativum plant of the presentinvention and at least one portion being a lab-grown material, preferably wherein the lab-grown material stems from a bacterial fermentation, or from a fungus, preferably from a 10filamentous fungus. Filamentous fungal biomass, usually named mycoproteins, is asuitable meat substitute since it is nutritious and has filaments and thus a texture perfectlymimicking meat fibrils. Regarding the type of residual water, nutrient supplementation,optimum conditions for biomass production, and characteristics of the mycoproteins, theoptimum growth condition can be at about pH of 4.5. Mycoprotein usually contains 19.44% 15(wt./wt.) protein with a high crude fiber content of 8.51% (wt./wt.) and a low fat content of1.56% (w/w). In addition, the amino acid and fatty acid contents are dominated by glutamicacid and polyunsaturated fatty acids, which are associated with an umami taste (Wikandariet al., 2023, https://doi.org/10.3390/molecules28030997). Particularly the physico-chemical and the viscoelastic properties of mycoproteins and the pea protein ingredients 20of the present invention allow that both sources of alternative proteins are easilycompounded and used together for food, particularly alternative meat or fish design, as thepI values of the major protein fractions and the viscoelastic properties are perfectly suitableto produce food with a high nutrition value and an excellent texture and stability.

In certain embodiments, depending on the nature of the ultimate product of interest 25comprising a pea protein ingredient, flour, protein concentrate, protein isolate, pea proteintexturate, pea protein powder, or a pea protein flake of a Pisum sativum plant, having analtered gelation pattern of the present invention, the product may include further additivesand ingredients. The skilled person being a food chemist, expert, a chemist, a cosmeticsengineer or a process engineer will well be in a position to adjust the final content of a 30product to achieve the optimum conditions guaranteeing easy and efficient manufacturingas well as a hygienically safe, food- or cosmetics-grade product with the right degree ofstability and non-perishable nature as needed.

In a sixth aspect, there is provided an alternative food or a cosmetic composition or product,or a hybrid composition or product, being selected from a solid and a semi-solid alternative 35 food or cosmetic composition or product, comprising at least one protein flour, a proteinconcentrate, a protein isolate, a pea protein texturate, a pea protein powder, or a peaprotein flake of a Pisum sativum plant claim of fourth aspect, or comprising at least onemixture of claim the fifth aspect, wherein the alternative food or an alternative cosmeticcomposition has an improved gelation in comparison to a reference material. 5 In one embodiment of the sixt aspect, the alternative food or the cosmetic composition orproduct, or the hybrid composition or product thereof has a pH in the range of about 3.5 toabout 8.5, preferably a pH from about 4.5 to about 8.

In certain embodiments, particularly for alternative food or hybrid food production andapplications, it is useful for the pea protein ingredient to be able to form a gel mass that 10can be sliced, shredded, crumbled, or spread. In view of the high gelation pattern of thepea protein ingredient, flour, protein concentrate, protein isolate, pea protein texturate, peaprotein powder, or the pea protein flake of a Pisum sativum plant of the present invention,these proteins are particularly suitable for use in preparing a gel mass with a certainstability. The ability of the gel mass to function in these applications depends on the 15viscoelastic properties of the pea protein ingredient itself, and/or in combination withadditional ingredients as needed for the desired consumable application. With the productsand compositions presented herein, and using the signature mutations as presented aboveand the agar ranking and vicilin to convicilin ratio providing a link between genetics andgelation properties, alternative and hybrid products and compositions can be prepared 20using the pea protein ingredients having an altered vicilin to convicilin ratio to influence thestability and rheology of the desired products based on the natural pea protein ingredientas stabilizer and texturing agent.

In certain embodiments, to form the stable gel mass, the pea protein ingredient, optionallyin combination with other ingredients to form a composition, is heated to the peak range of 25the DSC of the pea protein concentrate or the composition, preferably completely, asquickly as possible, followed by rapidly bringing the temperature down to below the DSCpeak range as soon as possible after reaching the maximum peak temperature. It isparticularly preferred to reach the peak end temperature of the denaturation peak of thecomposition to thus produce a stable gel mass with optimal properties. 30 Heating the pea protein ingredient or composition, generally to a temperature of around atleast 100°C, depending on the peak temperature range, is preferably performed in aclosable, pressure-tight vessel (pressure vessel) which is designed for a correspondingoverpressure resulting from the steam released during heating. he absolute pressure in the vessel may be between 1.3 and 5 bar, preferably between 2 and 3 bar. Upon heatingunder the counterpressure, the stable gel mass is formed, having a storage modulus G ′ ina plate-plate rheometer of at least 30,000 Pa, preferably at least 50,000 Pa, morepreferably between 50,000 Pa and 150,000 Pa, even more preferably between 50,000 Paand 100,000 Pa. 5 In a seventh aspect, there is provided a method of producing a protein flour, a proteinconcentrate, a protein isolate, a pea protein texturate, a pea protein powder, or a peaprotein flake of a Pisum sativum plant as defined in the fourth aspect above , or of a mixtureor composition as defined in the fifth aspect above, comprising the steps of: (i) providing atleast one pea from a Pisum sativum plant having an altered gelation pattern as defined in 10the first aspect above, or comprising at least one nucleotide molecule as defined in thesecond aspect above, or comprising at least one expression construct or vector as definedin the sixth aspect above; and (ii) optionally: dehulling the at least one pea; (iii) milling theat least one pea to obtain a flour; (iv) extracting at least one protein fraction, optionally: alsoincluding at least one starch fraction and/or at least one fat fraction, preferably wherein the 15extracting step is a dry extraction method, or wherein the extracting step is a wet extractionmethod; and (v) optionally: further processing and/or purifying the extracted protein fractionand thus obtaining an enriched and/or purified fraction selected from the group consistingof: protein flour, a protein concentrate, a protein isolate, a pea protein texturate, a peaprotein powder, or a pea protein flake of a Pisum sativum plant. 20 In certain embodiments of the seventh aspect, whole peas, with, but pereferably without astep of dehulling, may be put into an aqueous composition for fermentation followed bymilling the fermented peas, fractionating the fermented and milled peas so as to obtain atleast one protein comprising fraction and (d) isolating and optionally purigying pea proteinsfrom said at least one protein comprising fraction. Such a step of fermentation may be 25suitable to remove ono-, di-, and/or oligosaccharides and thus to enrich the protein fraction.

In an eigth aspect, there is provided a method of screening and/or selecting a Pisumsativum plant, wherein a pea protein ingredient obtained from said Pisum plant, preferablyas intermediate, has an altered gelation pattern as defined in the first aspect, or whereinsaid plant comprises at least one nucleotide molecule as defined in the second aspect, 30wherein the method comprises: (i) using at least one probe or at least one primer capableof identifying a signature mutation of the present invention; (ii) identifying and optionally:obtaining at least one Pisum sativum plant having at least one modification of a nucleotidesequence of at least one gene, including the regulatory region thereof, the gene encoding a convicilin protein, as defined in the first aspect, or a combination of more than onemodification.

In certain embodiments, suitable sets of PCR primers or probes, preferably labelledmolecular probes, can be designed using the signature mutations as position markers toscreen a germplasm for the presence or absence of at least one modification in a convicilin 5gene in comparison to a reference convicilin gene not carrying the relevant modification.

In a ninth aspect, there is provided a use of a protein flour, a protein concentrate, a proteinisolate, a pea protein texturate, a pea protein powder, or a pea protein flake of a Pisumsativum plant as defined in the fourth aspect, or a use of a mixture as defined in the fifthaspect for preparing an alternative food composition or an alternative cosmetic 10composition, preferably wherein the food composition is a solid or semi-solid alternativefood composition or an alternative cosmetic composition.

In certain embodiments, there is provided a protein flour, a protein concentrate, a proteinisolate, a pea protein texturate, a pea protein powder, or a pea protein flake of a Pisumsativum plant or a mixture or composition thereof for use as ingredient in an alternative 15food or cosmetic product or composition, optionally being a hybrid product or composition,wherein the composition may be an oral cavity care composition, particularly a cream, gel,ointment, dental gel or cream and the like. In view of the viscoelastic properties of theprotein compositions and isolates discloses herein, and further in view of the fact that peaconvicilin and variants thereof as disclosed herein can be produced in a sustainable way 20and represent products as such known to be safe for human use, the gelation propertiescan be used for cosmetic and further care compositions of the human or animal body asalternative to chemical substances in view of the favorable physico-chemical properties ofthe convicilin-containing material disclosed herein and its inherent tolerability and safety.

While several possible aspects are disclosed above, embodiments of the present invention 25are not so limited. These exemplary aspects are not intended to be exhaustive or tounnecessarily limit the scope of the invention, but instead were chosen and described inorder to explain the principles of the present invention so that others skilled in the art maypractice the invention. Indeed, various modifications of the invention in addition to thosedescribed herein will become apparent to those skilled in the art from the foregoing 30description. Such modifications are intended to fall within the scope of the appendedclaims.

The present invention is, in particular, captured by any one or any combination of one ormore of the above-mentioned aspects, with any other statement and/or embodiments.

The following Examples serve to provide further details on embodiments and enablementof the invention, but do not necessarily limit the scope of the invention.

Example 1: Bioinformatic analysis and selection and screening for suitable lines To verify the hypothesis that a targeted reduction of convicilin can indeed influence thegelation properties of pea protein ingredients in an adjustable manner, several in house 5lines and germplasm were analyzed and compared to sequences Pisum genomes ofCameor, further in-house lines / loci / germplasm, and genome assembly data, e.g. forZM6, were analyzed in detail (cf. Kreplak et al., 2019, and Yang et al., 2022, both supra).

Based on in silico predictions, the impact of the different types of pea storage proteins ongelation behaviour was assessed in a targeted way using a complex bioinformatic and 10biochemical analysis of various pea lines based on prior studies, and the following findingsof the inventors were made: ~ twenty pea lines were selected from the breeding collection,representing the greatest haplotype diversity in protein storage genes. From these lines,those which were able to produce a preferred pea protein are selected for furtherdevelopment. Selection criteria include but are not limited to pea plants producing peas 15with a lower convicilin content, a lower albumin content, or a combination of both properties.

Specifically, based on a detailed annotation, comparison and targeted mutagenesis of thepea reference genome data available, specific variants could be identified that altering theexpression of a convicilin gene, such as by mutation, deletion, or suppression ofexpression, it is possible to modulate the convicilin content of peas harvested from the 20cultivated plant. The levels of other storage proteins in peas may also be modulated by asimilar method.

Notably, as summarized in Tables 1 and 2 above, specific SNP mutations and certainmutations called "signature mutations" were identified that contribute either to a loss oftranscription, or a loss of translation of functional convicilin copies (usually two in a pea 25genome).

Based on these findings, and particularly the SNPs identified, TILLING populations can bescreened with the help of suitable primers and markers to identify the "convicilin low / highgelation +++" germplasm, especially that group having an almost abolished convicilinproduction, can be easily identified. Further, targeted gene editing can be used, e.g., using 30CRISPR/Cas technologies, by designing suitable gRNAs targeting the relevant convicilingene regions (coding / non-coding / upstream and downstream regulatory regions) toprovide pea germplasm producing total protein ingredients that have excellent gelation properties, which is usually a highly limiting factor for standard Pisum sativum pea proteinpreparations, making pea as valuable plant an even more interesting source for plant-based protein production.

Example 2: Production of pea flour from whole peas The material used was as follows: whole peas, any suitable variety, several kilograms; 5several bowls of adequate size for sorting; scoops and a brush; a Streckel&Schraderlaboratory dehuller LU-200; a Streckel&Schrader laboratory airleg separator LST1 and aultracentrifugal mill Retsch ZM200 with cyclone, attached bowl and 0,2mm sieve.

To obtain a pea protein flour, the following sample preparation was conducted: for thedehulling step, the whole peas were put into the dosing funnel of the dehuller. Using the 10dosing channel, the speed and amount of peas falling into the dehulling chamber can beadjusted. The slit between the dehuller discs can be adjusted to the size of the peas. Themix of dehulled whole and split peas and their hulls is collected in a drawer below the discsand is transferred into the dosing funnel of the airleg separator in order to sort them apart.The air flow to the different chambers therein can be adjusted with two screws. Peas that 15are still not dehulled, are transferred back to the dehuller while adjusting the slit accordingly.

Dehulled peas were then milled with the ultracentrifugal mill and the mentionedcomponents, carefully dosing them with a scoop. The resulting flour is collected in a bowl,and residues on parts of the mill are brushed off in order to clean them.

Example 3: Extraction of Pea Protein Isolate (PPI) from pea flour 20 To this end, the following material was used: pea flour (e.g., as produced in Example 2above; mixtures of flour from different peas or even of different plants can be used), 1MNaOH, 6M HCl, distilled water, 3L and 5L beakers, a lab scale PCE Instruments PCE-BSH10000, a propeller stirrer VWR VOS 40 digital, a magnetic stirrer IKA RH 2 basic, acentrifuge Eppendorf 5910 Ri cooled, with 4 centrifuge bottles 1L, a disperser/ 25homogenizer IKA ultraturrax T50 digital with S 50 N G45G disperser tool, a pH meterMettler Toledo FiveEasy F20, a spoon, silicone spatula and metal spatula, and a spraydryer Büchi S-300.

Pea flour was dispersed into distilled water while stirring with a propeller stirrer at around400 rpm in a ratio of S:L of 1:7, e.g. 500 g flour and 3000 mL water. The pH value of the 30dispersion was measured and adjusted using a pH-meter and 1M NaOH until it reachedpH 8. Extraction was conducted at 150 rpm at room temperature (at about 20 to 22°C) for 1 h. The dispersion was then transferred into the centrifugation bottles and centrifuged at4347 x g for 25 min to separate the dissolved protein-rich supernatant from the pellet richin starch and fibers. The pellet was disposed. The supernatant was transferred to the 3Lbeaker and stirred with the magnetic stirrer in order at 500 rpm to carefully dose the 6MHCL to precipitate the protein. The pH was adjusted to the isoelectric point of the proteins 5at 4.5, and the protein dispersion was stirred for 10 min to help the proteins agglomeratebetter. The dispersion was then centrifuged at 4347 x g for 25 min at 7 °C to lower theprotein solubility further and improve the protein yield. The resulting supernatant wasdisposed, and the protein pellet was carefully removed from the bottles with spatulas andtransferred into a beaker. The protein was washed with distilled water at a ratio of S:L of 101:5, using a ultraturrax disperser. The washed protein was again centrifuged underaforementioned conditions, and collected in a beaker. Neutralisation was carried out usingthe ultraturrax homogeniser, distilled water, until a protein concentration of about 10 % wasreached, and 1M NaOH. The neutralized protein solution was then spray-dried under thefollowing conditions: inlet temperature 130 °C, spraying gas flow 900 L/h, drying gas flow 15m³, product flow 6 mL/min. With these adjustable parameters the resulting outlettemperature was kept between 70-75 °C to ensure a safe product while maintaining thefunctionalities of native proteins as much as possible.

Specific parameters of this process are not intended to be limiting, and variations of thisextraction process by adjusting the pH, temperature and standing time, and other 20parameters may be utilized.

The standardized workflow from pea flour to pea isolate is shown in Figure 1 . Figure 2 ( Fig. 2A to E ) shows some working steps during the extraction process.

Figure 3 provides a detailed flow scheme for the Pea Protein Isolate (PPI) – ExtractionMethod: AE-IEP (Alkaline Extraction with Isoelectric Precipitation) as established. For this 25method, extraction at RT with a propeller stirrer is performed at 150 rpm, followed by proteinprecipitation on a magnetic stirrer at 400 rpm. This is followed by washing andneutralization, next by resuspension and homogenisation in distilled water with IKA ultraturrax T50. Finally, spray drying with Büchi S-300 is performed. Parameters: Inlet-Temp.130°C; Outlet-Temp. 70-75°C; Product flow: 6 mL / min; spraying gas: 900 L/h, drying gas: 3035m³/h. To this end, a stable PPI can be obtained.

Example 4: Protocol for sample preparation and storage protein ratio determination Ratio determination in all pea products was performed by CE-SDS (capillaryelectrophoresis with SDS buffer).

The material used was an Agilent ProteoAnalyzer, an Agilent Protein Broad Range P240Kit, a Vortex IKA VXR basic Vibrax, a centrifuge Heraeus Biofuge pico, a Pipette set(Eppendorf research 2-20 µl, 100-1,000 µl), a sodium dodecyl sulfate (SDS, ThermoScientific) PAGE done under reducing conditions using dithiothreitol (DTT) Serva.

Sample preparation was then performed as follows: for each experiment, 10 mg of each 5ground sample was mixed with 1 ml of a 1% SDS solution containing 0,4% dithiothreitol.The mixture was vortexed for 30 minutes to ensure thorough mixing and lysis of the sample.After vortexing the mixture was centrifuged at 15.000 g for 5 minutes to remove largeparticles and unbroken cells. Carefully, 4 µl of the supernatant was collected.

Sample analysis was subsequently performed by using the obtained supernatant, 10containing the solubilized proteins, which was prepared and analyzed using microfluidic gelelectrophoresis according to the manufacture´s assay guide. The electropherogram peakswere assigned to the corresponding protein fragments based on their molecular weight:gamma-vicilin (14–16 kDa), alpha-vicilin (19–20 kDa), basic legumin (22 kDa),beta+gamma-vicilin (25–30 kDa), alpha+beta-vicilin (36 kDa), acidic legumin (38–40 kDa), 15vicilin (48–51 kDa), convicilin (~75 kDa), and lipoxygenase (90–95 kDa).

Comparative measurements and visualization and quantification are shown in Fig. 4 and Fig. 5 , taken from the line KWS 1 ( Fig. 4 ), for line KWS 2 ( Fig. 5A to 5C ), and for Proklam( Fig. 5D to 5F ), all further detailed in Example 5 below. For line KWS 1 having a signaturemutation, a significantly higher vicilin to convicilin ratio was identified. In contrast, the ratio 20was lower for KWS 2 and for Proklam not carrying the signature mutation as detailed above.For Proklam, a total vicilin to convicilin ratio of 2.7:1 was identified.

Notably, KWS 1 was shown to carry signature mutations as shown in Tables 1 and 2 leading to a favorably altered gelation pattern as characterized by a total vicilin to convicilinration of ~ 5:1 that is much higher than the standard ratio of 2:1 to 4:1 defined as the 25standard ration present in a variety of pea lines (as average) not showing one of thesignature mutations of Table 1 and/or Table 2 in its genome. Interestingly, as for many pealines, it was confirmed that the average standard ratio of vicilin to convicilin is usuallybetween about 2:1 to about 4:1 (wt./wt.). This value was confirmed over a variety ofreference lines tested (average of about 100 lines and varieties available and screened). 30For our studies, lines were specifically screened for and/or generated that have a loweramount of convicilin, i.e., a ration shifted to vicilin until a complete knock-out of detectableconvicilin. These convicilin-low lines indeed showed a significantly enhanced gelationcorrelated to the decreased amount of convicilin present in the samples tested. Ground flour was used as reference as it represents the almost unprocessed pea protein containingsample after dehulling, yet it was also confirmed that the ratio of vicilin to convicilin wasalso detectable in a comparable ratio in further processed (PPI) samples (data not shown).

As further detailed below, the easy read-out parameter of a vicilin to convicilin ratio perfectlymatched with a further test established, an agar ranking test (see Example 5 ). With these 5both tests, an quick screening on "gelation-high" lines can be performed and theassociation with a signature mutation in a convicilin gene of the present invention can befurther tested and screened for.

Example 5: Gelling Test To establish a standardized test, the following appliances were used: a VorwerkTHERMOMIX TM6 with steamer attachment; a Microwave sharp inverter, a 100 mL Cups(e.g., Sarstedt), petri dishes (glass, diameter 6 cm); a silicone spatula, a metal spatula,Agar (Invitrogen, Select Agar, qualified for molecular genetics applications), a protein 5powder (protein powder can be used as a convenient and standardized equivalent in thetest established for all protein extracts, isolates and the like, in the present experiment aprotein isolate with a protein content of 85% was used).

Sample preparation for the gelation test was then performed as follows: 4 g protein powderwas stirred into 16 g water in a 100 mL Sarstedt Cup using a silicone spatula. Another set 10of samples were prepared for each protein powder with a dilution of 5 g protein powder andg water. Stirring was carried out until a homogeneous mass was obtained. The sampleswere then left to soak for one hour at room temperature (about 20°C to about 22°C). Duringthis time, the samples were thoroughly mixed after 30 min and again after 60 min.

Next, a steam treatment was performed. After the soaking phase, the samples were 15steamed using a Vorwerk Thermomix TM6. For this purpose, the samples were transferredto glass Petri dishes and placed in the steam cooking attachment of the appliance.Steaming was carried out for 35 min at 95°C. The samples were then cooled on ice for 15min.

Subsequently, the following steps were carried out for sampling and analysis: 1. photo 20documentation: the samples were photographed to record visual changes. 2. spatulasampling: A sample was taken with a spatula to check the consistency and homogeneity.3. spatula scratch sample: Another sample was taken by scratching with a spatula toassess the texture. 4. Documentation of optical attributes: The optical attributes of thesamples were recorded in detail. 5. agar equivalence test: Standards of 0; 1; 1.1; 1.2; 1.3; 251.4; 1.5; 1.6; 1.7 and 1.8 g/L were prepared from agar equivalent to the protein powdersample preparation. This dilution series was used to cluster the samples from 1 to 10 to betested against a uniform standard. The agar standard of 0 g/L corresponds to a value of 1,the agar standard of 1 g/L corresponds to a value of 2, the agar standard of 1.1 g/Lcorresponds to a value of 3, the agar standard of 1.2 g/L corresponds to a value of 4, the 30agar standard of 1.3 g/L corresponds to a value of 5, the agar standard of 1.4 g/Lcorresponds to a value of 6, the agar standard of 1.5 g/L corresponds to a value of 7, theagar standard of 1.6 g/L corresponds to a value of 8, the agar standard of 1.7 g/Lcorresponds to a value of 9, the agar standard of 1.8 g/L corresponds to a value of 10.

The results of the gelation and the associated optical and textural changes were carefullyrecorded and analyzed.

Table 3 KWS 1 KWS 2 KWS 3 Proklam (Commercially available) Dryingprocessspray-dried spray-dried spray-dried spray-driedColor of thegelOlive color Bright lightyellowBright lightyellowDark yellow, reddishFoambuildingThe bubbleshave spreadthroughout thegel.

The bubbleshave spreadthroughout thegel.

The bubbleshave spreadthroughout thegel.

Lass Foam, than theother gels, but alsospread throughout thegelGelling test%solid gelKWS 1,2,3 aresimilarsolid gel solid gel Moderately solid gel Stitch test 25%Puncture siterecognizable,puncture-proofPuncture siteflows shutagain, notpuncture-proof Puncture siterecognizable,puncture-proofNot puncture resistant,elastically deformable,liquid partScratch test%Clear boundary,similar to KWS 4Flowing backtogetherClear borderMost scratch-resistantFlowing back together,wateryScratch test%observationafter 24hours Bottom is clear,Scratch edgesare pretty straightScratch hole isclosing a little,Fluid is leakingon the bottomof the scratchhole Bottom isclear,Scratch edgesare falling alittle bit apart Flowing back together,watery Agar ranking 8 4 7 2 The results for the direct measurement and for the measurement after 24 h are shown in Figure 6 ( Fig. 6A to Fig. 6H ). By establishing the agar ranking test, a reliable and 5standardized test could be established that allows to classify pea material according to itsgelation pattern. Whereas KWS Proklam, for example, provides a rather liquid formulationwith a low gelation pattern, the different KWS lines KWS 1 to KWS 3, all showed a highergelation pattern, with KWS 3 as established and identified and carrying signature mutationhaving the strongest gelation of the compared gels. 10 Gel stability was positively correlated with a decrease of convicilin expressed. To this end,in addition to the total storage protein measurement allowing the determination of afavorable high gelation pattern (meaning a reduced amount of convicilin and thus anincreased amount of the ratio vicilin to convicilin), an easy agar ranking was establishedthat provides a visual read-out perfectly correlating with the amount of vicilin / convicilin 15identified on a molecular scale. As shown in Table 3 above, a scale from 1 to 10 wasestablished, 1 meaning almost liquid and 10 meaning very firm. Interestingly, the visually rather liquid (Proklam, Agar ranking 2) and not too stable in the test setting defined andconsistently used (Agar ranking 4, KWS 2) samples tested all came from lines not carryingthe favorable convicilin gene signature mutations as identified, introduced and tested forcertain lines. Further, the Agar ranking matched with the molecular results for the storageprotein test so that this test for screening lines quickly with a visual read-out before further 5molecular screening is applied could be provided.

KWS 1 and KWS 3, both showing a vicilin to convicilin ratio of ~ 5:1 and both carrying atleast one mutation (different pattern) in at least one convicilin gene, the mutation(s)representing signature mutations as identified (cf. Tables 1 and 2 herein above) alsoshowed a high Agar ranking score. 10 Our results suggest that genotype has influence on protein composition, as well as ontechnological-functional properties of pea proteins. This knowledge could be very useful inefforts to improve the quality of peas and pea protein products.

Strikingly, KWS line 1 and KWS line 3, both carrying signature mutations as identified tobe correlated to a higher gelation pattern both indeed showed a vicilin to convicilin ratio of 15about 5:1 (see Fig. 4 and 5 for KWS 1), whereas the reference line Proklam and a testlines KWS 2 and further lines tested in comparison, none of them having a signaturemutation in a convicilin gene identified to be highly suitable herein, showed a significantlylower Agar ranking score and – at the same time on molecular level – a much lower vicilinto convicilin (each total protein +/- signal peptide) ration of 2:1 to ~ 3.5:1 and 4:1 at most. 20 Example 6: DSC Analysis For testing certain parameters, differential scanning calorimetry (DSC) can be applied,particularly to check whether a concentrated pea protein solution is suitable for use as aprotein ingredient, and whether its proteins have a high or at least sufficient functionality.This test is thus useful in case the pea proteins with the favorably altered gelation pattern 25will be used together with other ingredients in compositions for alternative food, feed or forcosmetic purposes. Preferably, the denaturation enthalpy (which is the peak area of theDSC measurement) is usually at least 10 J/g protein, or more. To carry out the differentialcalorimetry measurement, at least 50 to 100 mg of a given pea protein concentrate samplewith a known protein content are weighed (use a steel vessel) in a volume of 100 μl and 30closed pressure-tight. Another steel vessel is then filled with water. This second vesselserves as a reference value for the measurement. One suitable device is that can be usedfor the measurement is the Mettler Toledo Type DSC 1 Star. of the devices performes atemperature scan with a heating rate of 2 K/min. The scan starts at 25° C and,preferably, ends at 130° C for a usual measurement. Denaturation of the proteins in a specifictemperature range are easily detacable in the DSC curve obtained as an endothermic peak,which is characterized by a peak start temperature, a peak maximum temperature, i.e., atemperature at the peak maximum, usually between 90° C and 125° C, and a peak endtemperature. The peak temperature range, which is passed through between the peak start 5temperature and the peak end temperature, is preferably between 25° C and 40° C, incertain settings between 30° C and 35° C. The peak area of the endothermic peak in theDSC curve is thus a measure of the extent of denaturation, as it is known to one skilled inthe art. Prticularly suitable concentrated plant protein solutionswill thus preferably be aboveJ/g protein, more preferably between 12 J/g and 30 J/g protein, whereinthe highest 10possible value is sought to be achieved.

Example 7: Oscillation Rheology To determine whether a concentrated pea protein isolate of this invention when in solutionhas sufficient aggregation behavior to function as a suitable protein ingredient for foodapplications, the pea protein concentrate solution is filled into a suitable steel vessel (e.g., 15beaker: C25 DIN system), usually between 10 and 15 ml. The vessel is then closedpressure-tight. The rheological properties can then be measured by means of the cylinder(C25 DIN system) which is positioned in the vessel containing the protein concentratesolution. Preferably, an absolutely pressure tight cylinder is used. Regarding relevantparameters, G ′ and G″ are measured, i.e., the elastic and the viscous portion of the 20viscoelastic concentrate, wherein it is well understood that the two portions G ′ and G ″change during the subsequent temperature cycles. The starting temperature is set to be25° C. Next, a rapid heating with a heating rate between 3 K/min and 5 K/min takes placeup to the relevant peak end temperature from the previous DSC measurement. Favorably,a short holding time step between 2 and 5 min at this temperature ensures that the pea 25protein concentrate or isolate was also completely exposed to this temperature.Subsequently, cooling is performed in a rapid way at a rate between about 3 K/min and 5K/min. G ′ and G ″ are continuously recorded. In the region of the peak initial temperaturefor suitable pea protein concentrate solutions with an aggregation behavior suitable orsufficient for carrying out the method according to the invention, a strong increase in the G ′ 30values is usually observed during the heating phase. The pea protein concentrate or isolatesolution is usually considered suitable for carrying out the method according to theinvention if the storage modulus still increases during the heating phase by at least 6x,preferably 6x-12x, more preferably 7x to 13x, compared with the storage value at thebeginning of the measurement, in particular at 25° C. The storage modulus G ′ will continue 35to increase during cooling until the gel is solidified. Again, the characteristics of aggregation behavior suitable for the methods according to the invention is the significant rise in G ′during the heating phase in the range between the peak start temperature and the peakend temperature.

Claims (15)

1. CLAIMS 1. A method of altering a gelation pattern of the pea protein ingredient of a Pisumsativum plant, the method comprising the following steps: (i) providing at least one pea comprising at least one nucleic acid moleculecomprising at least one modification of a nucleotide sequence of at least one gene,including the regulatory region thereof, wherein the at least one gene modifiedencodes a convicilin protein, wherein said at least one modification affects thegelation pattern of the pea protein ingredient, wherein said at least one modificationaffects at least one nucleotide sequence of a Psat06G0558100 reference gene (SEQID NO: 2) in comparison to the variant Psat06G0558100 reference gene (SEQ IDNO: 1) and/or of a Psat06G0558200 reference gene (SEQ ID NO: 66) in comparisonto the variant Psat06G0558200 reference gene (SEQ ID NO: 65), or of a combinationthereof; (ii) optionally: cultivating the Pisum sativum plant to obtain at least one peacomprising a protein composition having an altered gelation pattern in comparisonto a Pisum sativum plant not comprising the at least one modified convicilin gene, orthe regulatory region thereof; and (iii) obtaining a pea protein ingredient of said Pisum sativum plant, whereinsaidpea protein ingredient obtained, preferably as intermediate, has an alteredgelation pattern, wherein the altered gelation pattern is defined as an altered vicilinto convicilin ratio in a pea protein ingredient, preferably in a pea flour, in comparisonto a reference vicilin to convicilin ratio of 2:1 to 4:1 (wt./wt.) of a pea proteiningredient, preferably a flour, of a reference Pisum sativum plant, wherein the alteredvicilin to convicilin ratio is characterized by a decreased amount of convicilin incomparison to the amount measured for the reference Pisum sativum plant and thusan increased vicilin to convicilin ratio.

2. The method of claim 1, wherein the amount of convicilin in the pea proteiningredient, preferably in the pea flour, is reduced by at least 5%, at least 10%, atleast 15%, at least 20%, at least 25%, preferably at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 70%, morepreferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 100%, and most preferably almost completely lost in comparison to the amountmeasured for the reference Pisum sativum plant.

3. The method of claim 1 or 2, further comprising a step of processing, preferablyincluding a step of extraction, and/or purifying the pea protein ingredient of saidPisum sativum plant having an altered gelation pattern, wherein the processingpreferably at least includes a step of dehulling, to obtain a protein flour, a proteinconcentrate, a protein isolate, a pea protein texturate, a pea protein powder, or apea protein flake of a Pisum sativum plant.

4. The method of any of the preceding claims, wherein the convicilin protein hasa sequence selected from any one of SEQ ID NOs: 121 and 122, or a sequencehaving at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or at least 99% sequence identity thereto, and/or wherein the vicilinprotein has a sequence of SEQ ID NO: 123 or a sequence having at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%sequence identity thereto.

5. A method of any of the preceding claims, wherein the at least one modificationis a deletion, substitution, or an insertion, preferably a targeted substitution, of atleast one nucleotide sequence of the at least one gene, including the regulatoryregion thereof, encoding the convicilin protein.

6. The method of claim 5, wherein the at least one modification is provided by astep of mutagenesis, wherein mutagenesis includes chemical mutagenesis,radiation mutagenesis, and genome editing, wherein genome editing includes editingby site-directed nucleases, including zinc-finger nuclease (ZFNs) systems,transcription activator-like effector nuclease (TALENs) systems, meganucleasesystems and CRISPR/Cas systems, preferably wherein mutagenesis is resulting ina knock-down, more preferably a knock-out of at least one gene encoding convicilin.

7. A nucleic acid molecule comprising at least one modification of a nucleotidesequence of at least one gene, including the regulatory region thereof, the geneencoding a convicilin protein, wherein the modified nucleotide sequence is selectedfrom any one of SEQ ID NOs: 3 to 64 or 1, having at least one modification incomparison to the reference gene sequence of SEQ ID NO: 2, and/or wherein themodified nucleotide sequence is selected from any one of SEQ ID NOs: 67 to 120,or 65 having at least one modification in comparison to the reference gene sequenceof SEQ ID NO: 66, or any combination the aforementioned modifications of SEQ IDNOs: 3 to 64 and/or SEQ ID NOs: 67 to 120, or a nucleotide sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%sequence identity to the respective sequence provided that the respective signaturemutation as defined in Table 1 and Table 2 is still present, or a nucleic acid moleculecomprising at least one modification of a nucleotide sequence of at least one gene,including the regulatory region thereof, the gene encoding a convicilin protein,wherein the modified nucleotide sequence comprises at least one signature mutationas defined in Table 1 and Table 2 in comparison to the reference gene sequence ofSEQ ID NO: 2 or SEQ ID NO: 66, respectively.

8. An expression construct or a vector comprising at least one modifiednucleotide sequence of at least one convicilin gene and/or of the regulatory regionsthereof of claim 7.

9. A protein flour, a protein concentrate, a protein isolate, a pea protein texturate,a pea protein powder, or a pea protein flake of a Pisum sativum plant, having analtered gelation pattern, wherein the Pisum sativum plant comprises at least onemodification of a nucleotide molecule in a nucleotide sequence of at least oneconvicilin gene, including the regulatory sequence thereof, wherein the nucleotidemolecule is as defined in any one of claims 1 to 3 and 5 to 7, and/or wherein thePisum sativum plant, or a cell thereof, comprises at least one expression constructor at least one vector of claim 8, or wherein the Pisum sativum plant comprises atleast one modified convicilin protein of claim 4.

10. A pea protein mixture or a pea protein composition comprising a protein flour,a protein concentrate, a protein isolate, a pea protein texturate, a pea proteinpowder, or a pea protein flake of a Pisum sativum plant of claim 9 and additionallycomprising at least one further additive and/or ingredient.

11. An alternative consumable or a cosmetic composition or product, or a hybridcomposition or product, being selected from a solid and a semi-solid alternativeconsumable or cosmetic composition or product, comprising at least one proteinflour, a protein concentrate, a protein isolate, a pea protein texturate, a pea proteinpowder, or a pea protein flake of a Pisum sativum plant claim of 9, or comprising atleast one mixture of claim 10, wherein the alternative consumable or an alternativecosmetic composition has an improved gelation in comparison to a referencematerial.

12. The alternative consumable or the cosmetic composition or product, or thehybrid composition or product thereof of claim 11, wherein the composition orproduct has a pH in the range of about 3.5 to about 8.5, preferably a pH from about4.5 to about 8.

13. A method of producing a protein flour, a protein concentrate, a protein isolate,a pea protein texturate, a pea protein powder, or a pea protein flake of a Pisumsativum plant of claim 9, or of a mixture or composition of claim 10, comprising thesteps of: (i) providing at least one pea from a Pisum sativum plant having an alteredgelation pattern as defined in any one of claims 1 to 6, or comprising at least onenucleotide molecule of claim 7, or comprising at least one expression construct orvector of claim 8; and (ii) optionally: dehulling the at least one pea; (iii) milling the at least one pea to obtain a flour; (iv) extracting at least one protein fraction, optionally: also including at leastone starch fraction and/or at least one fat fraction, preferably wherein the extractingstep is a dry extraction method, or wherein the extracting step is a wet extractionmethod; and (v) optionally: further processing and/or purifying the extracted proteinfraction and thus obtaining an enriched and/or purified fraction selected from thegroup consisting of: protein flour, a protein concentrate, a protein isolate, a peaprotein texturate, a pea protein powder, or a pea protein flake of a Pisum sativumplant.

14. A method of screening and/or selecting a Pisum sativum plant, wherein a peaprotein ingredient obtained from said Pisum plant, preferably as intermediate, hasan altered gelation pattern as defined in any one of claims 1 to 6, or wherein saidplant comprises at least one nucleotide molecule of claim 7, wherein the methodcomprises: (i) using at least one probe or at least one primer capable of identifying asignature mutation of claim 7; (ii) identifying and optionally: obtaining at least one Pisum sativum planthaving at least one modification of a nucleotide sequence of at least one gene, including the regulatory region thereof, the gene encoding a convicilin protein, asdefined in any one of claims 1 to 7, or a combination of more than one modification.

15. A use of a protein flour, a protein concentrate, a protein isolate, a pea proteintexturate, a pea protein powder, or a pea protein flake of a Pisum sativum plant asdefined in claim 9, or a use of a mixture as defined in claim 10 for preparing analternative consumable composition or an alternative cosmetic composition,preferably wherein the consumable composition is a solid or semi-solid alternativeconsumable composition or an alternative cosmetic composition. Dr. Hadassa Waterman Patent Attorney G.E. Ehrlich (1995) Ltd. 35 HaMasger Street Sky Tower, 13th Floor Tel Aviv 6721407