Pea lines with improved taste profile

Abstract

The present invention relates to ingredients from a Pisum sativum line having desired organoleptic properties.

Description

[0001] TITLE: Pea lines with improved taste profile

[0002] FIELD OF THE INVENTION

[0003] The present disclosure relates to plant-based ingredients, particularly protein ingredients, suitable for use in food products, including vegetarian or vegan meat or dairy analogues. The plant-based ingredients are obtained from a novel pea line conferring an improved taste profile to its ingredients.

[0004] BACKGROUND OF THE INVENTION

[0005] Challenges resulting from continuous population growth, malnutrition and obesity, and environmental changes have caused a strong market interest in plant-based ingredients in the last years. Food producers are facing the challenges of providing plant-based food alternatives at large scale that meet consumers’ expectations in taste, texture, and other organoleptic properties.

[0006] To meet these demands, several sources of plant-based proteins have been considered by food producers, including but not limited to: soybeans; peas, chickpeas and split peas; lupin; hemp; potato; wheat; cranberries; beans such as navy, pinto, adzuki, fava, lima, black, red kidney, and mung beans; pumpkin seed and seed from other squash; and grains such as rice, sorghum, and millet.

[0007] One of the main limitations when working with plant-based ingredients, such as plant-based proteins, as an ingredient in food products is the perception of off-flavours. These off-flavours can be addressed to some extent using natural aromas and flavour-masking ingredients. However, the presence of certain aroma- and flavour-active compounds in plant-based isolates or concentrates render the product development process laborious, costly and time consuming, and further can be considered unfavourably by the consumer.

[0008] Peas as a plant-based ingredient source, especially a protein source, have attracted a great deal of interest from the food industry and consumers in recent years. Having an endogenous source of nitrogen lowers soil inputs needed for peas and is responsible for their high protein content, making peas an excellent source of plant proteins. Further, peas as high quality protein source have a much less carbon dioxide footprint (CO2 in g / 100 g protein produced) in comparison to meat-based protein sources or eggs (from chicken) (Sustainable Protein Sources. 1st Ed., 2016, Editors: Sudarshan Nadathur, Janitha P.D. Wanasundara, Laurie Scanlin, eBook ISBN: 9780128027769).

[0009] However, cultivars of Pisum sativum L. have been shown to differ greatly in their chemical composition, including their protein, fat and starch content. Both volatile and non-volatile compounds contribute to the sensory profile of peas and pea-based ingredients, based on their actual and relative concentrations.

[0010] Peas as a plant-based ingredient source, especially a protein source, have thus attracted a great deal of interest from the food industry and consumers in recent years. Having an endogenous source of nitrogen lowers soil inputs needed for peas and is responsible for their high protein content, making peas an excellent source of plant proteins. On top of that, compared to soybean or other proteins derived from plants, pea protein is associated with being more digestible and having relatively less allergenic responses and negative health controversies.

[0011] However, cultivars of Pisum sativum L. have been shown to differ greatly in their chemical composition, including their protein, fat and starch content. Both volatile and non-volatile compounds contribute to the sensory profile of peas and pea-based ingredients, based on their actual and relative concentrations (Eisner et al. (2021) Screening of twelve pea (Pisum sativum L) cultivars and their isolates focusing on the protein characterization, functionality, and sensory profiles 10 (4): 758)).

[0012] These differences in chemical composition require specialized processing methods to develop consumer desired food products, especially novel alternative food products, and parameters which may require batch-to-batch customization or continuous process adjustment, limiting the potential for pea ingredients to be produced or utilized at scale. Such differences also result in variations in ingredient and finished product attributes, including organoleptic properties such as taste or flavour, making the use of pea ingredients challenging in commercial food production.

[0013] One non-volatile compound found in peas and other plants is saponin, more specifically the group of saponin compounds. Saponins, including group A saponins (also termed herein as saponin A), group B saponins (also termed herein as saponin B), 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP)-saponins, group E saponins (also termed herein as saponin E), are amphiphilic glycosides of steroids and triterpenes and are known to cause “bitter”, “beany”, “grassy” and “astringent” flavours, which limits the use of saponin-containing plant-based ingredients in various food and other consumable applications. To overcome such limitations, it would be advantageous to enable the production of plant-based ingredients, such as proteins including pea proteins, having a lower or modified saponin content to reduce, substantially reduce, or eliminate the off-flavours and off-odours typically associated with such plant-based ingredients.

[0014] However, the plant saponin content fulfils an important defensive function in the plant, so that a complete knock out of key genes of the saponin pathway, which leads to elimination or significant reduction of total saponin content, might in some cases lead to reduced plant vigour, non-viable plants, or reduced plant yields, and might accordingly require the use of externally applied fungicides and other treatments to overcome this deficiency. In the field of food products, the use of fungicides and other treatments is not viewed favourably by food and feed producers and by end consumers. Therefore, a balance is needed between adapting plant-based ingredients for desired organoleptic properties while also providing solutions that provide a sufficient yield of plants and plant materials to be sustainable.

[0015] These differences in chemical composition require specialized processing methods and parameters which may require batch-to-batch customization or continuous process adjustment, limiting the potential for pea ingredients to be produced or utilized at scale. Such differences also result in variations in ingredient and finished product attributes, including organoleptic properties such as taste or flavour, making the use of pea ingredients challenging in commercial food production. There is therefore an ongoing need for pea ingredients, containing reduced off-flavours, improved taste properties and organoleptic attributes. Further, there is a great need in defining and creating suitable germplasm and pea lines that can be grown in a sustainable way and that harbour plant-protein ingredients of high consumable, particularly nutritional, value. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 (Fig. 1) shows a differential plot of Kameleon Peas and KWS076 (synonym: KM 17AE076, deposited under the Budapest Treaty as NCIMB 44401) peas in comparison as further detailed in Example 3 below. AR means aroma, FL means flavour, AT means aftertaste.

[0017] Figure 2 (Fig. 2) shows a differential plot of alternative milk beverage as further detailed in Example 4 below. AR means aroma, FL means flavour, AT means aftertaste.

[0018] BRIEF DESCRIPTION OF SEQUENCES

[0019] DEFINITIONS

[0020] As used herein, “a,” “an,” or “the” can mean one or more than one. For example, “a” cell can mean a single cell or a multiplicity of cells. Further, the term “a plant” may include a plurality of plants.

[0021] A “dry extraction method” includes protein extraction methods in which the protein fraction is obtained from the source material using air, pressure or other non-hydrating processes, or combinations thereof. A “wet extraction method” includes protein extraction methods in which the protein fraction is obtained from the source material using water or other solvents, such as acids, bases, combinations of such solvents, and the like. Preferably, the solvents used are food-grade or suitable for use in a food production facility.

[0022] As used herein, the term "flavour" means a quality that is detectable by taste and / or smell.

[0023] As used herein, the term "non-volatile flavour components" means molecules that provide flavour to a product, but are not volatile in the sense that they may not be removed by methods such as evaporation. The term applies to both liquids and solids.

[0024] As used herein, the expression “off-flavour” or “off-flavours” means an undesirable flavour, and refers to the complex interactions of various taste descriptors such as bitter, sour, earthy, beany, salty, sweet, green, savoury, nutty, umami, and the like. In addition, other organoleptic parameters such as dry, dull, powdery, moist, and the like play a role in flavour perception and are herein encompassed by the expression “off-flavours”.

[0025] As used herein, “plant-based ingredient” refers to any ingredient obtained from plants or plant parts in a processed form, including protein ingredients, soluble carbohydrate ingredients, such as starch, insoluble carbohydrate ingredients, such as fiber, lipid ingredients, ash ingredients, and the like. A plantbased ingredient may comprise any combination of proteins, carbohydrates, and lipids or ashes, etc., in any proportion. The term “plant-based ingredient” may herein be used interchangeably with the term “plant-based food ingredient”. As used herein, “protein ingredient,” “starch ingredient,” “carbohydrate ingredient,” “lipid ingredient” or “ash ingredient” includes any form of such ingredient suitable for use as-is or in combination with other components of a formula, such as the formula for a plant-based composition or a(n) (alternative) food product.

[0026] The plant-based ingredient may be in the form of an isolate, concentrate, flour, texturate, and the like.

[0027] “Protein texturate” or “textured protein” as used herein refers to an ingredient having a structural 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 other procedures used in the production of food for consumption. In general, textured proteins may be used to alter or enhance texture and bind water. Edible protein sources from 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 flour, textured soy concentrate, textured wheat protein, textured potato protein, or combinations thereof. Methods for protein texturization are known and described in the art, and may include, for example, high temperature and pressure extrusion, spinning, freeze texturization, chemical or enzymatic texturization, and the like.

[0028] “Protein flour” as used herein refers to an ingredient that contains milled peas.

[0029] An “alternative consumable product ingredient” as used herein refers to an ingredient from a plant, including a protein ingredient, which is suitable as part of a “consumable product” or of a “consumable composition”. Further, at least a part or fraction of the “alternative consumable product ingredient” is suitable as a substitute for a commonly known and commonly fabricated product ingredient, usually it is suitable as an alternative to, for example, animal-derived or animal-produced ingredients or food, including meat, eggs or dairy.

[0030] An “alternative food product” as used herein may be present in liquid form, in semi-liquid form or in solid form. As used herein, an “alternative food product” refers to a product for human or animal consumption that is usually made with ingredients from animal sources, but 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 include alternative beverages such as a milk substitute or a drinkable yogurt substitute, or alternative food products such as an egg, beef, dairy, poultry, or seafood substitute. Other alternative food products include pet or animal feed products in which some or all of the animal-sourced ingredients are substituted with plant-based ingredients. The replacement or substitution of the animal- sourced ingredient may in some embodiments be e.g. more than 70%, more than 80%, more than 90%, or more than 95%. In other embodiments, the replacement or substitution of the animal-sourced ingredient may be 70% or less, such as 60%, 50%, 40%, etc. Alternative food products may also include non-dairy beverages such as sports drinks or smoothies. Alternative food products may further refer to alternative nutritional products, such as plant-based powder, to be used as dietary or nutritional supplements.

[0031] An “alternative food” or “alternative nutrition” or “alternative (food / cosmetic) product I composition” as used herein thus refers to a food, including liquid food like beverages, which is usually a plant- or microorganism-based food that is an alternative to animal-derived food, including meat or dairy. Alternative food products may be of particular interest as an alternative source of proteins, but the term alternative food refers to any kind of nutritional building block, including proteins, carbohydrates, lipids, vitamins, minerals, fibers and the like that are suitable for food production and that are well accepted or even healthy as food and feed for human beings or farm animals and pets. An alternative food product or alternative food 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 product or an alternative composition as used herein, and any pea protein ingredient suitable for the production thereof of the present invention is specifically processed (industrially and / or mechanically and / or chemically and / or enzymatically) and a pea protein ingredient of this invention will usually be processed, isolated, concentrated and / or otherwise treated for inclusion in an alternative product or composition. Additionally a 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 product or composition, or of a mixture or hybrid product. In an alternative product or composition, the pea protein ingredient of this invention as alternative part or fraction of the alternative product or composition thus substitutes a part or fraction that would be present in 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 commonly known and commonly fabricated product.

[0032] 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.

[0033] 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 products without containing animal-based milk or being substantially free of animal-based products, and includes hybrid products made with lab-grown, fermented and animal-based components such as protein components. The dairy substitute may be completely free of any animal-based milk or animal-based milk protein or almost free of any animalbased milk protein, such as e.g. 90% free, or 95% free of any animal-based milk protein. The dairy substitute may be a dairy-free cheese, a dairy-free yogurt, a dairy-free ice cream, and the like.

[0034] As used herein, “meat substitute” or “meat substitute composition” or “meat alternative” refers to compositions that mimic the general, organoleptic, and / or nutritional properties of consumable products produced using any type of meat or meat analogue, including meat, fish, poultry, lab-grown and fermented meat products. This definition includes hybrid products made with lab-grown, fermented and animal-based components, such as protein components. A similar definition is used herein for “egg substitute”, “egg substitute composition,” and “egg alternative. ’’Whenever the terms “composition” or “pea protein composition” is used herein, it refers to a composition of any of the protein ingredients, flours, concentrates or isolates disclosed herein that are used in an extracted form together with other ingredients from different origins to provide said composition. An alternative food or a cosmetic is thus also a composition in this sense. In this context, a “mixture” or “pea protein mixture” is a specific form of a composition, wherein pea protein fractions from different peas or even from different plants or other sources are mixed with each other to provide a basic protein mixture (and optionally further additives or ingredients of different nature and / or origin) comprising pea protein of the present invention.

[0035] A “consumable product” as used herein refers to goods that are usually understood to be used up or depleted during normal business operations, such as food and beverage, office supplies, cleaning and sanitary products, and medical supplies. In line with the general understanding, there are two main types of consumables: durable consumables, which are expected to last over a long period of time, and non-durable consumables, which are expected to be used up relatively quickly. Examples of consumables include perishable foods and beverages, paper products, ink cartridges, cleaning chemicals, gloves, and syringes. The consumable products particularly dealt with herein are nondurable consumables that are non-toxic when swallowed or applied on the human or animal body based on their intended use that are usually made of at least one organic raw materials (and optionally others), including food, beverages, gels, ointments, tooth paste and the like. “Consumable products / composition” according to the present invention represent alternative products or compositions comprising at least one alternative plant-based ingredient, preferably a protein ingredient, according to the present invention.

[0036] A “gene” as used herein refers to the coding region of a gene, the non-coding region as well as upstream and / or downstream located regulatory sequences, including an enhancer, silencer, promoter elements (e.g., proximal, distal and core promoter elements), and 5' and / or 3'UTRs. Therefore, a modification of a gene may also include the modification of a non-coding and / or of a regulatory sequence thereof.

[0037] A “hybrid composition I product” or a “hybrid alternative (consumable, including food / cosmetic etc.) composition I product” as used herein refers to a product that at least partially comprises an “alternative food” or “alternative nutrition” or “alternative (food) product” comprising a plant-originating protein substance according to the present invention, but which may comprise further ingredients.

[0038] A “knock-down” of a gene refers to an experimental technique by which the expression of the gene (i.e., the transcription from DNA to RNA and thus the amount of active RNA transcripts) is reduced. A reduced expression can e.g., be achieved by gene silencing reducing or abolishing the transcription rate and thus decreasing the amount of functional RNA available.

[0039] 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 abolished at all or (on protein level) to such an extent that nearly no functional protein is expressed, or is expressed very level at or about the detection limit. This can e.g., be achieved by replacing or interrupting the sequence of the target gene. For example, an early or premature additional stop codon, preferably close to the start codon can lead to a premature transcription stop that results in the complete loss of functionally translated protein. Alternatively, a “knock-out” can be achieved by a mutation leading to a variation in the naturally occurring splice donor or splice acceptor site of a eukaryotic gene comprising exons and introns. The splice donor site usually includes an almost invariant sequence GU at the 5' end of the intron, within a larger, less highly conserved region. The splice acceptor site 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 result in 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 a functional knock-out in the sense that the sequence encoded by a gene is no longer transcribed or translated. A “knock-out” may also be produced by a deletion of a gene, or of a substantial part thereof, on a genomic level, optionally accompanied by a substitution against another sequence. A “mutation” or a “genome modification” in the context of the present invention refers to any change of a coherent nucleic acid sequence by modifying a nucleic acid sequence at a given nucleotide sequence position that results in at least one difference in the (nucleic acid) sequence distinguishing it from the original sequence. In particular, a modification can be achieved by insertion or addition of one or more nucleotide(s), or substitution or deletion of one or more nucleotide(s) of the original sequence or any combination of these.

[0040] A “nucleic acid construct”, “construct” or “expression construct” refers to a nucleic acid molecule encoding or comprising one or more genetic elements, which upon introduction into 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 regulatory sequences such as promoter and terminator sequences facilitating expression of the genetic elements) as well as spacers and introns. The genetic elements of the present invention can also be encoded on a set of constructs, which constructs can be introduced into a cell simultaneously or consecutively.

[0041] The term "RNAi" or “RNA silencing” or “gene silencing” as used herein interchangeably refer to the process called RNA interference meaning a gene down-regulation (or knock-down) mechanism meanwhile demonstrated to exist in all eukaryotes. The mechanism was originally recognized and described in plants where it was called "post-transcriptional gene silencing" or "PTGS". In RNAi, small RNAs function to guide specific effector proteins to a target nucleotide sequence by complementary base pairing resulting in degradation of the target. A “gene silencing construct” or “RNAi agent” usually comprises so called “sense” and “antisense” sequences. Sense and antisense sequences are complementary sequences, which are present in reverse orientation in a nucleic acid sequence. If a nucleic acid construct comprises a sense and a corresponding antisense sequence, the two complementary sequences form an RNA double strand upon transcription, which results in an “RNA hairpin”. In an RNA hairpin, sense sequences and corresponding antisense sequences, together form a double strand and are separated by an “intervening intron loop sequence” forming the loop of the hairpin structure.

[0042] The terms Pisum sativum (L.) plant and pea plant, and short only pea, are used interchangeably 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.

[0043] A “(pea) plant ingredient” as used herein is to be understood as the total amount of protein that can be extracted from a (pea) plant fruit or seed (dry or fresh). A “(pea) protein ingredient” is in turn to be understood as the total amount of protein of the (pea) plant.

[0044] The term “protein concentrate” is a protein ingredient with a concentration of about 30% to 60%. A “protein isolate” is an even more concentrated protein ingredient with a concentration of about 60% to about 100%. A “protein flour” represent the protein that can be obtained directly after dehulling and milling. As this protein flour is not yet heavily processed, it reflects the original content of protein ingredients rather directly. Therefore, the “protein flour” was also used to define standard ratios by the inventors when comparing different material herein below. A “protein texturate” or “texturized vegetable protein” is used to describe a usually further defatted flour product that is particularly suitable and used as a meat analogue or meat extender. It is quick to cook, with a protein content comparable to some meats. The terms “protein flake” or “protein powder” further describe the form of the protein. A “protein powder” is usually composed of fine, dry particles produced by the grinding, crushing, or disintegration of a solid substance.

[0045] The term “vector” refers to an element used for introducing a nucleic acid construct or set of nucleic acid constructs into a cellular system. The vector may be a plasmid or plasmid vector, cosmid, artificial yeast artificial chromosomes (YAC), bacterial artificial chromosome (BAC) or P1 artificial chromosomes (PACs), phagemid, bacterial phage based vector, a modified viral vector, an Agrobacterium shuttle vector, an isolated single-stranded or double-stranded nucleic acid sequence, comprising DNA and RNA sequences in linear or circular form, or a mixture thereof, for introduction or transformation into a plant, plant cell, tissue, organ, or material according to the present disclosure.

[0046] 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, and derivatives and progeny thereof. Plant cells include without limitation, for example, cells from seeds, from mature and immature cells or organs, including embryos, meristematic tissues, seedlings, callus tissues in different differentiation states, leaves, flowers, roots, shoots, male or female gametophytes, sporophytes, pollen, pollen tubes and microspores and protoplasts etc.

[0047] “Mutagenesis” refers to a technique, by which modifications or mutations are introduced into a nucleic acid sequence in a random or non- site-specific way. For example, mutations can 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-specific modifications”, on the other hand, rely on the action of site-specific effectors such as nucleases, 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 specific location within the target sequence.

[0048] A “protein composition” as used herein refers to a protein isolate directly obtainable from a fruit, seed, particularly from a specific pea of the genus Pisum, or to a flour, a protein fraction, a purified or partially purified protein fraction. The protein composition, depending on the way of preparing the same, may include denatured and / or partially fragmented proteins, as the proteins may have undergone denaturation and / or fragmentation during thermal, chemical and / or mechanical processing / purification. A protein composition may consist of substantially one protein or a fragment thereof, particularly convicilin, or it may be a protein composition mixture comprising other proteins, particularly globulins and further pea proteins.

[0049] “TILLING” (Targeting Induced Local Lesions in Genomes) is a process, which allows to identify 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 for performing TILLING are known to the skilled person.

[0050] A “functional homolog(ue)” as used herein refers to a molecule having substantially the same function as a reference molecule. A “structural homolog” refers to a homolog having a substantial degree of sequence identity to a reference molecule, or a part thereof it originates from.

[0051] The term "nucleotide sequence" in relation to the present invention includes genomic DNA, cDNA, synthetic DNA, and RNA. Preferably it means DNA, more preferably genomic DNA.

[0052] Amino acids are referred to herein using the name of the amino acid, the three-letter abbreviation or the single letter abbreviation. The term “protein", as used herein, includes proteins, polypeptides, and peptides. As used herein, the term “amino acid sequence” is synonymous with the term “polypeptide” and / or the term “protein”. In some instances, the term “amino acid sequence” is synonymous with the term “peptide”. In some instances, the term “amino acid sequence” is synonymous with the term “enzyme”.

[0053] A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), betabranched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted non-essential amino acid residue in a protein of the invention is preferably replaced with another amino acid residue from the same side chain family.

[0054] Conservative amino acid substitutions may occur over the full length of the sequence of a polypeptide sequence of a functional protein such as an enzyme. In one embodiment, such mutations are not pertaining the functional domains of an enzyme. In one embodiment, conservative mutations are not pertaining the catalytic centers of an enzyme.

[0055] In the present disclosure and claims, the conventional one-letter and three-letter codes for amino acid residues may be used. The three-letter code for amino acids as defined in conformity with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.

[0056] A plant line being “homologous” or a “homologous line” to KWS076 as used herein refers to a line that has been created by modifying or introducing at least one gene locus of KWS076 based on the present disclosure to modify a gene in the saponin pathway in a targeted way in line with the present disclosure based on the knowledge and teaching provided for KWS076. Further, also orthologous genes to the ones identified herein can be derived and used for providing plants with an optimized saponin biosynthesis pathway, e.g. in other plant of the family of Fabaceae, formerly Leguminosae (used interchangeably herein).

[0057] Whenever the present disclosure relates to the percentage of identity of nucleic acid or amino acid sequences to each other, or to the identity of a homologue, orthologue, or paralogue of a given gene locus to another, these values define those values as obtained by using the EMBOSS Water Pairwise Sequence Alignments (nucleotide) programme (www.ebi.ac.uk / Tools / psa / emboss_water / ) nucleic acids or the EM-BOSS Water Pairwise Sequence Alignments (protein) programme (www.ebi.ac.uk / Tools / psa / emboss_water / ) for amino acid sequences. Alignments or sequence comparisons as used herein refer to an alignment over the whole length of two sequences compared to each other. Those tools provided by the European Molecular Biology Laboratory (EMBL) European Bioinformatics Institute (EBI) for local sequence alignments use a modified Smith-Waterman algorithm (see www.ebi.ac.uk / Tools / psa / and Smith, T.F. & Waterman, M.S. “Identification of common 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 open penalty = 10 and gap extend penalty = 0.5 or (ii) for nucleic acid sequences: Matrix = DNAfull, gap open penalty = 10 and gap extend penalty = 0.5. The skilled person is well aware 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 original organism a molecule originates from.

[0058] “Protein Percent” or “Starch Percent” is generally measured by NIR spectrophotometry, but can be measured by other means well-known in the art.

[0059] As used herein, the term "plant" includes whole plants, including descendants or progeny thereof. As used herein unless clearly indicated otherwise, the term "plant" intends to mean a plant at any developmental stage. The term "plant part" includes any part or derivative of the plant, including particular plant tissues or structures, plant cells, plant protoplast, plant cell or tissue culture from which plants can be regenerated, plant calli, plant clumps and plant cells that are intact in plants or parts of plants, such as seeds, kernels, cobs, flowers, cotyledons, leaves, stems, buds, roots, root tips, stover, and the like. Plant parts may include processed plant parts or derivatives, including flour, oils, extracts, protein fractions, etc. "Parts of a plant" are e.g., shoot vegetative organs / structures, e.g., leaves, stems and tubers; roots, flowers and floral organs / structures, e.g., bracts, sepals, petals, stamens, carpels, anthers and ovules; seed, including embryo, endosperm, and seed coat; fruit and the mature ovary; plant tissue, e.g., vascular tissue, ground tissue, and the like; and cells, e.g., guard cells, egg cells, pollen, trichomes and the like; and progeny of the same. Parts of plants may be attached to or separate from a whole intact plant. Such parts of a plant include, but are not limited to, organs, tissues, and cells of a plant, and preferably seeds. A "plant cell" is a structural and physiological unit of a plant, comprising a protoplast and a cell wall. The plant cell may be in form of an isolated single cell or a cultured cell, or as a part of higher organized unit such as, for example, plant tissue, a plant organ, or a whole plant. "Plant cell culture" means cultures of plant units such as, for example, protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes and embryos at various stages of development. "Plant material" refers to leaves, stems, roots, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant. This also includes callus or callus tissue as well as extracts (such as extracts from taproots) or samples. A "plant organ" is a distinct and visibly structured and differentiated part of a plant such as a root, stem, leaf, flower bud, or embryo. "Plant tissue" as used herein means a group of plant cells organized into a structural and functional unit. Any tissue of a plant in planta or in culture is included. This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue culture and any groups of plant cells organized into structural and / or functional units. The use of this term in conjunction with, or in the absence of, any specific type of plant tissue as listed above or otherwise embraced by this definition is not intended to be exclusive of any other type of plant tissue. In certain embodiments, the plant part is a plant organ, tissue, or cell. In certain embodiments, the plant part is seed, pollen, oocyte, protoplast, inflorescence, embryo, or callus.

[0060] As used herein, a “control plant” or “control plant part” or “control cell” or “control seed” refers to a plant or plant part or plant cell or seed that has not been subject to the methods and compositions described herein. A “control” or “control plant” or “control plant part” or “control cell” or “control seed” provides a reference point for measuring changes in phenotype of the subject plant or plant cell. A control plant or plant cell may comprise, for example: (a) a wild-type plant or cell, i.e., of the same genotype as the starting material for the genetic alteration which resulted in the subject plant or cell; (b) a plant or plant cell of the same genotype as the starting material but which has been transformed with a null construct (i.e. with a construct which has no known effect on the trait of interest, such as a construct comprising a marker gene); (c) a plant or plant cell which is a non-transformed segregant among progeny of a subject plant or plant cell; (d) a plant or plant cell genetically identical to the subject plant or plant cell but which is not exposed to conditions or stimuli (e.g., sucrose) that would induce expression of the gene of interest; or (e) the subject plant or plant cell itself, under conditions in which the gene of interest is not expressed. In certain instances, a control plant of the present disclosure is grown under the same environmental conditions (e.g., same or similar temperature, humidity, air quality, soil quality, water quality, and / or pH conditions) as a subject plant described herein. Similarly, a control protein or control protein composition can refer to a protein or protein composition that is isolated or derived from a control plant. In specific embodiments, a control plant, plant part, or plant cell is a plant, plant part, or plant cell that does not have a mutated nucleotide sequence in a gene or a regulatory region of a gene as disclosed in the present invention.

[0061] "Progeny” includes an F1 pea plant produced from the cross of two pea plants where at least one plant includes a pea line, variety, or cultivar represented by a sample of seeds which have been deposited under the terms of the Budapest Treaty as NCIMB 44401 , or crossed therewith, and progeny further includes, but is not limited to, subsequent F2, F3, F4, F5, F6, F7, F8, F9, and F10 generational crosses with the recurrent parental line. As used herein with respect to a parameter, the term “decreased” or “decreasing” or “decrease” or “reduced” or “reducing” or “reduce” or “lower” or “loss” refers to a detectable (e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%) negative change in the parameter from a comparison control, e.g., an established normal or reference level of the parameter, or an established standard control. Accordingly, the terms “decreased”, “reduced”, and the like encompass partial reduction compared to a control. As used herein, the terms “eliminating” or “eliminated” or “eliminate” encompass a complete reduction, or an almost complete reduction compared to a control. For example, it refers to a 96%, 98% or 100% negative change in the parameter from a comparison control.

[0062] As used herein, the terms “modification” or “modify” or any other variations of the term refers, in the context of saponin content or expression of saponin-related genes, to a modulation of the total saponin content by varying the saponin profile, as described in greater detail below. For example, if saponin B levels are reduced, with a concomitant increase in DDMP saponin levels the total saponin content does not significantly change, but instead the saponin profile is modified as a result of modifications made to the expression of saponin-related genes.

[0063] As used herein with respect to a parameter, the term “increased” or “increasing” or “increase” refers to a detectable (e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, 120%, 150%, 200%, 300%, 400%, 500%, or more) positive change in the parameterfrom a comparison control, e.g., an established normal or reference level of the parameter, or an established standard control. Accordingly, the terms “increased”, “increase”, and the like encompass a mild, moderate, or significant increase compared to a control.

[0064] DETAILED DESCRIPTION OF THE INVENTION

[0065] The present inventors have detected, characterized and further developed a novel pea line conferring a particularly good taste profile to its ingredients and alternative food products produced therefrom.

[0066] In a first aspect, there is provided a plant-based ingredient, preferably a protein-ingredient, of, obtained from or obtainable from a pea of a Pisum sativum, wherein a representative sample of seeds of said plant was deposited under NCIMB No. 44401 , wherein the plant-based ingredient provides an improved taste profile as compared to a plant-based ingredient from a pea from a reference Pisum sativum plant, wherein the improved taste profile is due to at least one signature mutation in at least one gene, preferably a non-coding region of the gene, wherein the gene is involved in the saponin biosynthesis pathway.

[0067] In one embodiment, the signature mutation in at least one gene involved in the saponin biosynthesis pathway is in a gene, or the non-coding region thereof, encoding a certain enzyme or protein directly involved in the saponin biosynthesis pathway. In another embodiment, the signature mutation is part of the germplasm deposited under NCIMB No.

[0068] 44401 , wherein the mutation directly or indirectly influences the saponin biosynthesis pathway.

[0069] In one embodiment of the first aspect, the plant-based ingredient, preferably a protein-ingredient, of, obtained from or obtainable from a pea of a Pisum sativum, wherein a representative sample of seeds of said plant was deposited under NCIMB No. 44401 , carrying at least one signature mutation can be identified by at least two markers, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or at least twelve markers, wherein said markers are individually selected from a combination of markers of SEQ ID NO: 10 to 21 , or a modified marker thereof provided that the modified marker still comprises or encodes the relevant SNP signature mutation of a plant deposited under NCIMB No. 44401 at the position indicated with an ambiguity symbol in SEQ ID NO: 10 to 21 , respectively, as shown and explained in Table 6 for the material deposited under NCIMB No. 44401 , i.e., wherein the relevant genotype position is at nucleic acid number 101 with respect to SEQ ID NO: 10 to 21 , respectively, and wherein the nucleic acid at the relevant genotype position is: A, if the marker is a modified marker of SEQ ID NO 10; G, if the marker is a modified marker of SEQ ID NO 11 ; C, if the marker is a modified marker of SEQ ID NO 12; C, if the marker is a modified marker of SEQ ID NO 13; C, if the marker is a modified marker of SEQ ID NO 14; T, if the marker is a modified marker of SEQ ID NO 15; A, if the marker is a modified marker of SEQ ID NO 16; T, if the marker is a modified marker of SEQ ID NO 17; G, if the marker is a modified marker of SEQ ID NO 18; G, if the marker is a modified marker of SEQ ID NO 19; T, if the marker is a modified marker of SEQ ID NO 20.

[0070] In another embodiment, at least one signature mutation is identified by at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or eight markers, wherein said markers are individually selected from a combination of markers of SEQ ID NO: 32 to 38, or a modified marker thereof provided that the modified marker still comprises or encodes the relevant SNP signature mutation (position indicated with an ambiguity symbol in SEQ ID NO: 32 to 38), respectively, as shown in Table 7, preferably wherein a BAS specific marker according to SEQ ID NO: 32 is used, i.e., wherein the relevant genotype position is at nucleic acid number 101 with respect to SEQ ID NO: 10 to 21 , respectively, and wherein the nucleic acid at the relevant genotype position is: T, if the marker is a modified marker of SEQ ID NO 32; A, if the marker is a modified marker of SEQ ID NO 33; C, if the marker is a modified marker of SEQ ID NO 34; T, if the marker is a modified marker of SEQ ID NO 35; T, if the marker is a modified marker of SEQ ID NO 36; T, if the marker is a modified marker of SEQ ID NO 37; T, if the marker is a modified marker of SEQ ID NO 38.

[0071] Notably, the combined use of markers of SEQ ID NO: 10 to 21 , or of modified markers thereof as defined above, and of markers of SEQ ID NO: 32 to 38, or of modified markers thereof, allows both the precise identification of signature mutations being characteristic for NCIMB No. 44401 and at the same time being characteristic for the improved taste profile of the relevant plant-based ingredient obtained from or obtainable from said pea, or from other Pisum sativum material carrying the relevant signature marker pattern as identified in Tables 6 and / or 7 below alone or in combination characteristic of the relevant Pisum sativum material according to the present invention.

[0072] Preferably, according to the various aspects and embodiments disclosed herein, the plant-based ingredient does not contain genetic information and is not capable of self-replication or being reproduced in a biological system.

[0073] As each of the markers of SEQ ID NO: 10 to 21 comprises at least one characterizing signature mutation or single-nucleotide polymorphism (SNP) as indicated with a IUPAC ambiguity symbol at the relevant genotype position as explained in the below Examples, said signature mutations being characteristic for NCIMB No. 44401 , these signature mutations allow the precise identification of the relevant plant germplasm, wherein the plant germplasm results in a plant-based ingredient with an improved taste profile as compared to a plant-based ingredient from a pea from a reference Pisum sativum plant.

[0074] A “marker” or a combination of markers as used herein may refer to any one of SEQ ID NO: 10 to 21 , wherein a marker may also be a “modified marker”, or a combination of more than one modified marker, originating from and based on SEQ ID NO: 10 to 21 provided that the modified marker still comprises the relevant signature mutation or SNP at the relevant genotype position indicated with an ambiguity symbol in SEQ ID NO: 10 to 21 , respectively, discriminating the material unambiguously from other germplasm. Such a modification of a marker may thus comprise a shortening or a prolongation of the marker, as long as the core sequence of at least 10 nucleotides up- and downstream of the respective signature mutation or SNP are still present. The skilled person is well aware of the development of, for example, KASP markers as one example of modified markers that can be custom made based on the marker information as provided herein depending on the screening or selection method of interest.

[0075] In one additional aspect, there is provided a method of screening and optionally selecting a plant as basis for obtaining from said plant a plant-based ingredient, preferably a protein-ingredient, of, obtained from or obtainable from a pea of a Pisum sativum, wherein a representative sample of seeds of said plant was deposited under NCIMB No. 44401 , wherein the plant-based ingredient provides an improved taste profile as compared to a plant-based ingredient from a pea from a reference Pisum sativum plant, wherein the improved taste profile is due to at least one signature mutation in at least one gene, preferably a non-coding region of the gene, wherein the gene is involved in the saponin biosynthesis pathway, wherein the screening and optionally selecting uses at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve markers, wherein said markers are individually selected from a combination of markers of SEQ ID NO: 10 to 21 , or a modified marker thereof provided that the modified marker still comprises the relevant SNP signature mutation at the genotype relevant position indicated with an ambiguity symbol in SEQ ID NO: 10 to 21 , respectively and as shown in Table 6.

[0076] Pisum sativum KWS076 has shown uniformity and stability, as described in the following description information. Pisum sativum KWS076 has been self-pollinated a sufficient number of generations with careful attention to uniformity of plant type and has been increased with continued observation for uniformity.

[0077] Pisum sativum KWS076 has the following morphologic and other characteristics based primarily on field data collected in Wetze, Germany. The characteristics of KWS076 are described in the following:

[0078] Maturity: Number of Nodes at first bloom: 13

[0079] Height: KWS076 is about 70 cm high and is about 5 cm taller than KWS Exam.

[0080] Vine:

[0081] Habit determinate

[0082] Branching 1-2 Branches

[0083] Internodes Zig Zag

[0084] Stockiness medium

[0085] Number of Nodes 13

[0086] Leaflets:

[0087] Color Green

[0088] Wax Light

[0089] Marbled Yes

[0090] No. of leaflet pairs Two

[0091] Stipules missing

[0092] Flower color:

[0093] Venation Greenish

[0094] Standard White

[0095] Wing White

[0096] Keel White

[0097] Pods :

[0098] Shape Slightly Curved

[0099] Color Green

[0100] Surface Rough

[0101] Length 7 cm

[0102] End Blunt

[0103] Width 8 mm (Between Sutures)

[0104] No. of Seeds per Pod 5

[0105] Peas:

[0106] Color Yellow

[0107] Seeds (Dry, Mature):

[0108] Shape Round

[0109] Surface Smooth

[0110] Color Pattern Monocolor

[0111] Primary Color Yellow

[0112] Hilum Floor Color Light, same color as primary color Cotyledon Color Yellow

[0113] Grams per 100 Seeds 17

[0114] In one aspect, there is provided a Pisum sativum plant or seed, preferably for producing a plant-based ingredient as disclosed herein, or a composition comprising at least one of said plant-based ingredients,, wherein said plant or seed is or is a progeny of a seed deposited under NCIMB No. 44401 , preferably by selfing or as a first generation offspring, preferably wherein the at least one Pisum sativum plant or seed is characterized by at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or at least twelve markers, wherein each marker identifies a SNP signature mutation, wherein said markers are individually selected from a combination of markers of SEQ ID NO: 10 to 21 , or a modified marker thereof provided that the modified marker still comprises or encodes the relevant SNP signature mutation at the relevant position indicated with an ambiguity symbol in SEQ ID NO: 10 to 21 , respectively, as shown in Table 6, and / or wherein the at least one Pisum sativum plant or seed is characterized by at least one signature mutation identified by at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or eight markers, wherein said markers are individually selected from a combination of markers of SEQ ID NO: 32 to 38, or a modified marker thereof provided that the modified marker still comprises or encodes the relevant SNP signature mutation as shown in Table 7, preferably wherein a BAS specific marker according to SEQ ID NO: 32 is used.

[0115] In another aspect, there is provided the use of a Pisum sativum plant or seed for producing a plantbased ingredient as disclosed herein, or a composition comprising at least one of said plant-based ingredients, wherein said plant or seed is or is a progeny of a seed deposited under NCIMB No. 44401 , preferably wherein the at least one Pisum sativum plant or seed is characterized by at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or at least twelve markers, wherein each marker identifies a SNP signature mutation, wherein said markers are individually selected from a combination of markers of SEQ ID NO: 10 to 21 , or a modified marker thereof provided that the modified marker still comprises or encodes the relevant SNP signature mutation at the relevant position indicated with an ambiguity symbol in SEQ ID NO: 10 to 21 , respectively, as shown in Table 6, and / or wherein the at least one Pisum sativum plant or seed is characterized by at least one signature mutation identified by at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or eight markers, wherein said markers are individually selected from a combination of markers of SEQ ID NO: 32 to 38, or a modified marker thereof provided that the modified marker still comprises or encodes the relevant SNP signature mutation as shown in Table 7, preferably wherein a BAS specific marker according to SEQ ID NO: 32 is used.

[0116] In one embodiment, there is provided a plant-based ingredient, preferably a protein-ingredient, of the first aspect, wherein the pea plant comprises at least one signature mutation(s) of at least one nucleotide sequence of a gene affecting saponin biosynthesis pathway in the plant in comparison to a reference (synonymously: control) Pisum sativum plant, wherein the gene encodes and is selected from a cytochrome P450 monooxygenase (P450), a uridine diphosphate-dependent (UDP) glycosyltansferase (UGT), a TSAR transcription factor, a basic leucine zipper (bZIP) transcription factor, or a squalene synthase, including a cytochrome P450 monooxygenase (P450), a uridine diphosphatedependent glycosyltansferase (UGT), and / or a Triterpene Saponin Biosynthesis Activating Regulator (TSAR) transcription factor, including TSAR1 , wherein the mutation reduces, modifies or eliminates the biosynthesis of saponin in the plant or a plant part in which it is expressed, optionally wherein the plant comprises at least one further mutation affecting the taste profile in a gene involved in a pathway other than the saponin pathway, preferably in a lipoxygenase (LOX) biosynthesis pathway.

[0117] The present inventors could surprisingly identify a specific genetic link between mutation genes, including the non-coding and regulatory regions thereof, affecting the saponin pathway or a LOX pathway in a way that reduces undesired off-flavours in peas so that the flavor of a plant-based, preferably a protein-ingredient, of a pea carrying at least one signature mutation of the present invention makes the respective pea line much more favorable for applications as an ingredient in an alternative food.

[0118] Lipoxygenase (also: LOX EC 1 .13.11.12; linoleate: oxygen oxidoreductase) is an iron containing dioxygenase catalyzing the hydroperoxidation of linoleic acid and other polyunsaturated fatty acids and their esters, as well as glycerides containing a cis,cis-1 ,4-pentadiene double bond system, originating a hydroperoxide that possesses a chain with a cis, trans conjugated double bond (Grechkin, Recent developments in biochemistry of the plant lipoxygenase pathway; Progress in Lipid Research; 1998). It is known that the action of lipoxygenase generates very reactive compounds like free radicals, which can react with chlorophyls, carotenoids, ascorbic acid, phenols and also a-tocopherol (vit. E) (Gomboeva, Shumaev, Gessler, & Lankin, 2001). LOX plays an important role in forming biologically active substance, as it is engaged in the biosynthesis of controllers such as jasmonic acid, his methyl ester, abescisic acid, traumatic acid, which plays an important role in growth of plants and response to biotic and abiotic stress (Babitha et al; Purification and properties of lipoxygenase induced in downy mildew resistant pearl millet seedlings due to infection with Sclerospora graminicola' Plant Science; 2004).

[0119] Products formed during the enzymatic reaction are very important for food quality. Hydroperoxides derivatives and products of their degradations can react with proteins, peptides and amino acids, which can result in off-flavours production and lowering nutritional values of the products.

[0120] Consequently, KWS076 is a highly interesting line, as it harbors valuable signature mutations in genes involved in the saponin and / or the lipoxygenase pathway both known to be relevant for controlling off- flavours associated with plant-based ingredients and with the sensory and taste profile of products and compositions made thereof.

[0121] In a further embodiment, there is provided a plant-based ingredient, preferably a protein-ingredient, wherein the improved taste profile is independently selected from the group consisting of an improved flavour profile, including a less bitter flavour, a less sour flavour, a less beany flavour and / or a less pea- like flavour, an improved mouthfeel, including a less powdery mouthfeel, a less dull mouthfeel and / or a less coating mouthfeel, a less bitter aftertaste, an improved aroma, including as a less pea-like aroma and / or a less green / grassy aroma, or any combination thereof.

[0122] Closely related reference or control Pisum sativum varieties to the Pisum sativum plant of the present invention are KWS Kameleon, KWS Exam and KWS Flam. Both varieties are commercially available from KWS Momont SAS, France, and / or from KWS SAAT SE & Co. KGaA in Einbeck, Germany. Further, the ZM6 reference genome (cf. Yang, T., Liu, R., Luo, Y. etal. “Improved pea reference genome and pan-genome highlight genomic features and evolutionary characteristics,” Nat Genet 54, 1553- 1563; 2022) can be used as reference.

[0123] Seeds of a plant the Pisum sativum line KWS076 synonymously called KM 17AE076 disclosed herein and recited in the appended claims have been deposited with the National Collections of Industrial, Food and Marine Bacteria (hereinafter, “NCIMB”), Wellheads Place, Dyce, Aberdeen, AB21 7GB, Scotland, Great Britain, 27 June 2024 and have received accession number NCIMB 44401 (cf. Form BPM). The material was confirmed to be viable by NCIMB as confirmed 27 June 2024 (cf. Form BP / 9). NCIMB is a recognized International Depository Authority under the Budapest Treaty. This deposit of KWS076 seeds was made under the Budapest Treaty and will be maintained in the NCIMB depository for at least the enforceable life of the patent, and will be replaced if the deposit becomes nonviable during that period. Additionally, Applicant has satisfied all the requirements of 37 C.F.R. Sections 1 .801-1 .809, including providing an indication of the viability of the sample.

[0124] By performing a deep screen and bioinformatic comparison, the present inventors could identify that KWS076 carries several signature mutations, i.e., single-nucleotide polymorphism (SNP) mutations present in the KWS076 line, but not in a reference line, namely as aligned with to the pea reference genome “ZW6” that has been described in the literature, for example in Yang, et al. 2022, supra).

[0125] Mutations in genes, including the coding and the non-coding, for example, the regulatory, regions in KWS076 were identified and confirmed in KWS076 as providing a genetic basis for the improvement in sensory performance. While not intending to be bound by theory, it is believed that these one or more mutations resulted in an alteration in the saponin profile in the peas of KWS076, and the resulting protein ingredient from the peas demonstrated an improved sensory profile as compared to protein from a commercial pea variety.

[0126] In one embodiment according to the various aspects disclosed herein, there is provided at least one mutation in the coding region of at least one gene of the saponin and / or the LOX pathway in a Pisum plant the creating an early stop codon in the coding sequence that has a favourable effect on the saponin and / or jasmonic acid and / or abscisis acid and / or traumatic acid profile, wherein an early stop codon may be preferred. Other mutations in other saponin or LOX pathway genes may also play a factor in altering the saponin and / or jasmonic acid and / or abscisis acid and / or traumatic acid profile and resulting the improved sensory performance of a pea protein ingredient obtained from KWS076. In a further embodiment of the first aspect above, the plant-based, preferably pea-based, ingredient is a protein, a starch, a fat or combination thereof, preferably a protein.

[0127] In a second aspect, there is provided a plant-based composition comprising a plant-based ingredient of any embodiment of the first aspect above.

[0128] In specific embodiments, the plant-based composition may further comprise (i) a second plant-based ingredient from a plant different from the first plant-based ingredient; and / or (ii) a protein ingredient obtained from an animal source, from a microbe, a fungi, fermentation and / or cell culture to obtain a hybrid composition.

[0129] In yet another embodiment of the second aspect, the composition is a protein composition.

[0130] In a third aspect, there is provided an alternative consumable product comprising a plant-based ingredient or a plant-based composition according to any embodiment of the first and second aspect above.

[0131] In one embodiment, the alternative product is selected from an alternative beverage product, including an alternative dairy beverage, an alternative non-dairy beverage, or an alternative fermented beverage, or wherein the alternative product is selected from an alternative consumable product, including an alternative dairy product, an alternative seafood product, an alternative egg product and an alternative meat product.

[0132] In yet another embodiment, the alternative consumable is a vegetarian or a vegan product.

[0133] The seed from pea line KWS076, the plant produced from the seed, the hybrid pea plant produced from the crossing of the variety with any other pea plant, hybrid seed, and various parts of the hybrid pea plant can thus be utilized for obtaining plant-based products. Industrial uses include but are not limiting to, human food, livestock feed, and as a raw material in industry.

[0134] As used herein the term “plant product” will be understood to mean the product derived from or produced by a plant of pea line KWS076, for example, the tissues or structures of the plant of KWS076, such as the flower, fruit, seed, leaves, stems etc., produced by the plant. Further, the pea seeds produced from or derived from KWS076 can be crushed, or a component of the pea seeds can be extracted, in order to comprise a plant extract such as protein concentrate, protein isolate, texturate, meal, flour, and for a food or feed product. In other embodiments, a processed plant product includes, but is not limited to: dehydrated, cut, sliced, ground, pureed, dried, baked, fried, canned, jarred, washed, brined, packaged, refrigerated, frozen and / or heated pods, and / or seeds of the pea plants of the invention, or any other part thereof. In further embodiments, a processed plant product includes a protein, sugar or other carbohydrate, fiber, and / or aromatic compound that is extracted, purified or isolated from pea plants disclosed herein. In embodiments, the processed plant product includes washed and packaged pods and / or seeds (or parts thereof) of KWS076, for example, in a canned or frozen form. In other embodiments, the processed plant product is a whole pod that has been dehydrated and / or baked. In certain embodiments, peas from KWS076 can be used to produce a pea meal or flour. Pea meal or flour produced from KWS076 can also be used to produce a pea protein concentrate, a pea protein isolate, and any other form of ingredient obtained or extracted from the peas.

[0135] In a further embodiment, peas from KWS076 can be used to produce various types of “fillers” in food products. Examples of food products containing pea derived products are protein powder, meat-free burgers / minced meat / sausages, dairy alternative drinks, yogurt alternatives, vegan cheese and puffs, and protein bars. Thus, the peas from KWS076 can be processed to produce a texture and appearance similar to many other foods.

[0136] Additionally, in certain embodiments, the peas from KWS076 can be used to produce an intermediate, preferably a protein-based ingredient intermediate, suitable as additive or filler in a cosmetic composition or product.

[0137] According to the various embodiments herein related to a protein-based ingredient from KWS076, or a homologous line thereof, the high protein content of the peas having a favourable taste profile is particularly advantageous.

[0138] For consumption, i.e., for food and feed, peas from KWS076 can be favourably used according to the various aspects and embodiments provided herein to produce edible protein ingredients which offer a healthier replacement for animal protein in alternative meats, alternative dairy products, beverages, nutritional supplements, and the like. Peas are approximately 21.2-32.9% protein, 36.9%-49% starch, and 14-26% dietary fiber, by dry weight (for reference values, cf., e.g., Geerts, M. E. J., et al, “Mildly refined fractions of yellow peas show rich behaviour in thickened oil-in-water emulsions,” Innovative Food Science and Emerging Technologies. (2017). 41 : 251-258; Lan, Y., et al., “Solid dispersion-based spray-drying improves solubility and mitigates beany flavour of pea protein isolate,” Food Chemistry. (2019). 278: 665-673; Pietrasik, Z., et al., “Utilization of pea starch and fibre fractions for replacement of wheat crumb in beef burgers,” Meat Science. (October 2019). 161 : 107974).

[0139] Peas being a legume crop have two aspects that distinguish them from most other food crops. Firstly, they are rich with macro and micronutrients: being a good source of protein (rich with essential amino acids as tryptophan and lysine), slowly digestible carbohydrates, B group vitamins, minerals, dietary fiber (soluble and insoluble), phytosterols, and a-linolenic, acid. They also provide some amounts of squalene, tocopherols, polyphenols and triterpenic acids.

[0140] Compared to soybean or other proteins derived from plants, pea protein is associated with being more digestible and having relatively less allergenic responses and negative health controversies.

[0141] However, as described above, there remain challenges with using pea protein as a food ingredient, particularly at scale, due to organoleptic performance issues that can be overcome with a pea line of KWS076, or a homologous line. In certain embodiments of providing a further processed, purified and / or isolated plant-based protein material, the ingredient is in the form of a concentrate, an isolate, a texturate, a powder, a flake, or a flour, or the ingredient is a protein, a soluble carbohydrate, an insoluble carbohydrate, a lipid, an ash, or a combination thereof, preferably a protein.

[0142] In yet another embodiment, the plant or plant part the ingredient is obtained from is selected from a crop, including soybean (Glycine max), beans (Phaseolus spp.), common bean (Phaseolus vulgaris), fava bean (Vicia faba), mung bean (Vigna radiata), pea (Pisum sativum), chickpea (Cicer arietinum), peanut (Arachis hypogaea), lentils (Lens culinaris, Lens esculenta), lupins (Lupinus spp.), white lupin (Lupinus albus), mesquite (Prosopis spp.), carob (Ceratonia siliqua), tamarind (Tamarindus indica), alfalfa (Medicago sativa), barrel medic (Medicago truncatula), birdsfood trefoil (Lotus japonicus), licorice (Glycyrrhiza glabra), clover (Trifolium spp.), oat, chickpea, kidney bean, corn, potato, wheat, sunflower, canola or rice, preferably wherein the plant or plant part the ingredient is obtained from is a legume (family of Fabaceae, formerly Leguminosae), more preferably pea (Pisum sativum) soybean (Glycine max), beans (Phaseolus spp.), including common bean (Phaseolus vulgaris), and lupins (Lupinus spp.), including white lupin (Lupinus albus).

[0143] In a fourth aspect, there is provided a method of producing an alternative consumable product the third aspect, comprising (i) providing a plant-based ingredient or a plant-based composition of any one of first or second; ii) adding at least one further additive and / or ingredient; (iii) producing an alternative consumable product.

[0144] In a fifth aspect, there is provided a use of a plant-based ingredient or composition according to any one of the first or second aspect for reducing or eliminating off-flavours in an alternative consumable product, preferably in an alternative consumable product as defined in the third aspect, preferably for providing an alternative food, feed or cosmetic composition, optionally in the form of a hybrid product.

[0145] In a sixth aspect, there is provided a method of producing a plant-based ingredient of the first aspect, comprising the steps of: (i) providing a seed from the Pisum sativum plant having a representative sample of seeds deposited under NCIMB No. 44401 , or optionally of a progeny thereof; and (ii) extracting a fraction, including a protein fraction, a starch fraction or a fat fraction, from the pea.

[0146] In one embodiment of the sixth aspect, at least one mutation representing a signature mutation, or more than one signature mutation forming a signature mutation pattern in at least one gene in the saponin and / or the LOX biosynthesis pathway as identified in the Pisum sativum plant having a representative sample of seeds deposited under NCIMB No. 44401 can be introduced by mutagenesis or genetic engineering, wherein mutagenesis includes chemical mutagenesis, radiation mutagenesis, and genome editing wherein genome editing includes editing by site-directed nucleases, including zinc- finger nuclease (ZFNs) systems, transcription activator-like effector nuclease (TALENs) systems, meganuclease systems and CRISPR / Cas systems; and (ii) extracting a fraction, including a protein fraction, a starch fraction or a fat fraction, from the pea. In one embodiment, the method produces a protein ingredient in the form of a concentrate, an isolate, a texturate, a powder, a flake, or a flour.

[0147] In a further embodiment of the method of the sixth aspect, the extracting step is a dry extraction method or a wet extraction method.

[0148] In yet another embodiment, at least one signature mutation or a signature mutation pattern may be introduced into another plant, wherein the plant or part of the plant is selected from a crop, including soybean (Glycine max), beans (Phaseolus spp.), common bean (Phaseolus vulgaris), fava bean (Vicia faba), mung bean (Vigna radiata), pea (Pisum sativum), chickpea (Cicer arietinum), peanut (Arachis hypogaea), lentils (Lens culinaris, Lens esculenta), lupins (Lupinus spp.), white lupin (Lupinus albus), mesquite (Prosopis spp.), carob (Ceratonia siliqua), tamarind (Tamarindus indica), alfalfa (Medicago sativa), barrel medic (Medicago truncatula), birdsfood trefoil (Lotus japonicus), licorice (Glycyrrhiza glabra), clover (Trifolium spp.), oat, chickpea, kidney bean, corn, potato, wheat, sunflower, canola or rice, preferably wherein the plant or plant part the ingredient is obtained from is a legume (family of Fabaceae, formerly Leguminosae), more preferably pea (Pisum sativum) soybean (Glycine max), beans (Phaseolus spp.), including common bean (Phaseolus vulgaris), and lupins (Lupinus spp.), including white lupin (Lupinus albus).

[0149] A "site-directed nuclease" herein refers to a nuclease or an active fragment thereof, which is capable of specifically recognizing and cleaving DNA at a certain location, the target sequence. Such nucleases typically produce a double-strand break (DSB), which is then repaired by non-homologous end-joining (NHEJ) or homologous recombination (HR). Site-specific nucleases include meganucleases, homing endonucleases, zinc finger nucleases, transcription activator-like nucleases and CRISPR nucleases, or variants including nickases or nuclease-dead variants thereof.

[0150] A "CRISPR nuclease", as used herein, is a specific form of a site-directed nuclease and refers to any nucleic acid guided nuclease which has been identified in a naturally occurring CRISPR system, which has subsequently been isolated from its natural context, and which preferably has been modified or combined into a recombinant construct of interest to be suitable as tool for targeted genome engineering. Any CRISPR nuclease can be used and optionally reprogrammed or additionally mutated to be suitable for the various embodiments according to the present invention as long as the original wild-type CRISPR nuclease provides for DNA recognition, i.e., binding properties. CRISPR nucleases also comprise mutants or catalytically active fragments or fusions of a naturally occurring CRISPR effector sequences, or the respective sequences encoding the same. A CRISPR nuclease may in particular also refer to a CRISPR nickase or even a nuclease-dead variant of a CRISPR polypeptide having endonucleolytic function in its natural environment. A variety of different CRISPR nucleases / systems and variants thereof are meanwhile known to the skilled person and include, inter alia, CRISPR / Cas systems, including CRISPR / Cas9 systems (EP2771468), CRISPR / Cpf1 systems (EP300951 1 B1), CRISPR / C2C2 systems, CRISPR / CasX systems, CRISPR / CasY systems, CRISPR / Cmr systems, CRISPR / MAD systems, including, for example, CRISPR / MAD7 systems (WO2018236548A1) and CRISPR / MAD2 systems, CRISPR / CasO systems (Pausch et al., Science, 2020, 10.1126 / science.abb1400), CRISPR / CasZ systems and / or any combination, variant, or 30 catalytically active fragment thereof. A nuclease may be a DNAse and / or an RNAse, in particular taking into consideration that certain CRISPR effector nucleases have RNA cleavage activity alone, or in addition to the DNA cleavage activity.

[0151] The "guide molecule" or "guide nucleic acid sequence" (usually called and abbreviated as guide RNA, crRNA, crRNA+tracrRNA, gRNA, sgRNA, depending on the corresponding CRISPR system representing a prototypic nucleic acid-guided site-directed nuclease system), which recognizes a target sequence to be cut by the nuclease. The at least one "guide nucleic acid sequence" or "guide molecule" comprises a "scaffold region" and a "target region". The "scaffold region" is a sequence, to which the nucleic acid guided nuclease binds to form a targetable nuclease complex. The scaffold region may comprise direct repeats, which are recognized and processed by the nucleic acid guided nuclease to provide mature crRNA. A pegRNAs may comprise a further region within the guide molecule, the so- called "primer-binding site". The "target region" defines the complementarity to the target site, which is intended to be cleaved. A crRNA as used herein may thus be used interchangeably herein with the term guide RNA in case it unifies the effects of meanwhile well-established CRISPR nuclease guide RNA functionalities. Certain CRISPR nucleases, e.g., Cas9, may be used by providing two individual guide nucleic acid sequences in the form of a tracrRNA and a crRNA, which may be provided separately, or linked via covalent or non-covalent bonds / interactions. The guide RNA may also be a pegRNA of a Prime Editing system. The at least one guide molecule may be provided in the form of one coherent molecule, or the sequence encoding the same, or in the form of two individual molecules, e.g., crRNA and tracr RNA, or the sequences encoding the same.

[0152] In certain embodiments, a donor plant or donor plant population, comprising one or more alleles 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 any plant of interest, to introduce one or more alleles associated with an improved trait in the recipient plant or plant population, e.g. as part of a breeding program. The method according to the present invention may be used to identify one or more progenies of such crossings, having an improved trait.

[0153] In certain embodiments, the plants, preferably the peas, produced by these selected plant, preferably pea, lines or varieties have a lower or modified saponin content, and therefore lower off-flavour perception, as compared to peas from plants in which the expression of one or more of the biosynthetic pathway genes is not reduced, significantly reduced, or eliminated. The selected pea plants are cultivated, and the peas may be harvested from these plants. The protein ingredient can be extracted from the harvested peas, resulting in a pea protein ingredient having reduced off-flavours as compared to a control (or reference) pea protein ingredient made from peas in which the expression of one or more of these genes is not altered (hereinafter referred to as “control peas”). In certain aspects and embodiments of the method of the sixth aspect, the provision of at least one Pisum sativum plant seed having a saponin and / or LOX biosynthesis pathway associated gene locus showing at least one signature mutation, or a signature mutation pattern as present in the genome of a Pisum sativum plant having a representative sample of seeds deposited under NCIMB No. 44401 in comparison to a reference or control Pisum sativum plant not carrying the at least on can be achieved by using a reference or control plant not carrying the at least one signature mutation or the signature mutation pattern can be achieved transiently and temporarily by reducing the transcription of a gene in the saponin and / or LOX biosynthesis pathway, e.g., via using knock down RNAi construct, to achieve a phenotype of a reduced off-flavour of the present invention. Details of the invention are further described in the following non-limiting examples.

[0154] EXAMPLES

[0155] Example 1 : Sensory evaluation of peas: set-up

[0156] Sensory evaluations were conducted on peas, protein isolates from peas, and alternative milk beverages containing such pea protein isolates. The sensory dimensions included appearance, aroma, flavour, mouthfeel, and aftertaste, and each attribute is summarized in Table 1 :

[0157] TABLE 1

[0158] Dimension Attribute from to Definition

[0159] Colour Intensity bright dark Measuring the colour intensity of the sample.

[0160] Appearance Particles (on glass) little many Measuring the amount of particles on the glass.

[0161] Foam little much Measuring the foam of the product.

[0162] Overa .l.l . Int.ens .it.y little . int .en Measuring the overall intensity of the product,

[0163] 3se inc .lud ,.ing all percept ..ions.3 r

[0164] Pea little intense Measuring the intensity of the pea aroma.

[0165] Cereal little intense Measuring the intensity of the cereal aroma.

[0166] Aroma „ ..... . . , M,easur . .. . .

[0167] Green / Grassy little intense ingathe . i .nte ,nsity3of the g • rassy and / or green

[0168] 3aroma, associated with mowed? gras3s.a

[0169] Sweetish little intense Measuring the intensity of the sweetish aroma.

[0170] Sourly little intense Measuring the intensity of the sourly aroma.

[0171] Overa .l.l . Int.ens .it.y . l.it.t..le . int .en Measuring the overall intensity of the product,

[0172] 3se inc .lud ..ing all percept ..ions.3 r

[0173] Pea little intense Measuring the intensity of the pea flavour.

[0174] Cereal little intense Measuring the intensity of the cereal flavour.

[0175] G „reen / Grassy . . Measuring the intensity of the grassy and / or green3little intense f ,,lavour, aassoc .iat .ed , wit3h mowed grass.

[0176] Flavour Milky little intense Measuring the intensity of the milky flavour (cow

[0177] Sweet little intense Measuring the intensity of sweet taste.

[0178] Sour little intense Measuring the intensity of sour taste.

[0179] Bitter little intense Measuring the intensity of bitter taste.

[0180] Salty little intense Measuring the intensity of salty taste.

[0181] Powdery little intense Measuring the intensity of the powdery mouthfeel.

[0182] Dull little intense Measuring the intensity of the dull mouthfeel.

[0183] Texture Coating little intense Measuring the intensity of the coating mouthfeel.

[0184] Astringent little intense Measuring the intensity of the astringent mouthfeel.

[0185] Viscosity thin thick Measuring the intensity of the moisture mouthfeel.

[0186] Overa .l.l . Int.ens .it.y . l.it.t..le . int .ense Me .as ,u. ring the overall intensity of the product,

[0187] 3including all ..3 r perceptions.

[0188] Pea little intense Measuring the intensity of the pea aftertaste.

[0189] Cereal little intense Measuring the intensity of the cereal aftertaste

[0190] A .f.t.ert .ast .e r B,.i*tt.er little . int .ense Measuring the intensity of the bitter aftertaste (30 second .s) ..

[0191] Sweet little intense Measuring the intensity of sweet aftertaste.

[0192] Sour little intense Measuring the intensity of sour aftertaste. r B,.i*tt.er little . int.ense Measuring the intensity of the bitter aftertaste (60 second ,s .). 1

[0193] The differences in various attributes were analyzed for statistical significance based on an Analysis of Variance (mixed ANOVA), which determines whether products are significantly discriminated based on an attribute. To perform the mixed ANOVA, an anchor product is a fixed factor, and the tested products are the random factors, which provides the estimated means and the “least significant difference” (hereinafter, “LSD”). The LSD corresponds to the minimal required difference expressed as a scale distance between two products to conclude that these are significantly different (Fischer’s LSD 95%). The “descriptor discrimination” (hereinafter “DD”) indicates how many times the LSD “fits” within the highest and lowest intensity scores occurring among the products. If the DD is greater than 1 , the attribute is significantly discriminating between the products.

[0194] Example 2: Sensory evaluation of peas: testing

[0195] 20g of cooked and mashed peas obtained from pea line KWS076, having a representative sample of seeds deposited under NCIMB No. 44401 , were evaluated for sensory properties in comparison to cooked and mashed peas from a commercial pea variety Flam and Kameleon, available from KWS SAAT SE & Co. KGaA in Einbeck, Germany. A trained sensory panel evaluated the peas for flavour, mouthfeel / texture and aftertaste (data not shown, triplicate measurements not yet finished, first data very promsing). The preliminary data available at date indicate an improved mouthfeel and taste, and a reduced overall bitterness and bitter aftertaste, attributed to the peas from KWS076 as compared to peas from the control Kameleon or Flam variety.

[0196] Example 3: Sensory evaluation of protein isolate extracted from peas

[0197] 50g of protein isolate powder from peas obtained from pea line KWS076, having a representative sample of seeds deposited under NCIMB No. 44401 , were evaluated for sensory properties in comparison to a protein isolate obtained from a commercially available pea variety Kameleon. A trained sensory panel evaluated 5% protein isolate solutions made from combining the protein isolate powders with 1 ,000ml water. The results of the evaluation are shown in Table 2 and Figure 1.

[0198] TABLE 2: Mean Score Analysis: Protein isolate

[0199] The results of the Mean Score analysis indicate reductions in the aroma, flavour and aftertaste attributes listed in Table 1 for the protein isolate extracted from peas from KWS076 as compared to a protein isolate extracted from peas from the commercial Kameleon variety. The results of the Mean Score analysis indicate reductions in the aroma, flavour and aftertaste attributes listed in Table 2 for the protein isolate extracted from peas from KWS076 as compared to a protein isolate extracted from peas from the commercial Kameleon variety. The result of this comparison are additional shown in Figure 1.

[0200] Example 4: Sensory evaluation of yly alternative milk beverage

[0201] 200g of a protein isolate as described in Example 2 were added to 8,000 mL of a commercial alternative milk base formula provided by VF Nutrition GmbH, doing business as vly (registered trademark), of Berlin, Germany. A trained sensory panel of 10 participants evaluated the alternative milk beverage for sensory properties including the aroma, flavour, and aftertaste attributes listed in Table 3 and shown in Figure 2 as compared to the same alternative milk base formula made with a protein isolate from the commercially available Flam variety.

[0202] TABLE 3: Mean Score Analysis: Alternative milk beverage

[0203] The results of the Mean Score analysis comparing the statistically significant differentiating attributes are shown in graphical form in Fig. 2. These data indicate a significantly improved aroma and flavour, and a reduced and less intense bitter aftertaste, attributed to the peas from KWS076 as compared to peas from the commercially available Flam variety.

[0204] While several possible embodiments are disclosed above, embodiments of the present invention are not so limited. These exemplary embodiments are not intended to be exhaustive or to unnecessarily limit the scope of the invention, but instead were chosen and described in order to explain the principles of the present invention so that others skilled in the art may practice the invention. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0205] Example 5: Saponin determination in pea products by LC-MS MS

[0206] To verify that the KWS076 line indeed has a favorable saponin profile, comparative Liquid Chromatography with MS / MS (MRM detection) (short: LC / MS) were performed.

[0207] For LC-MS / MS, 100 mg of each ground sample was mixed with 10 mL of 70% ethanol:30% DI water solution. The mixture was vortexed for 30 seconds followed by ultrasonic extraction for 30 minutes. The mixture was vortexed for 30 seconds again followed by centrifugation for 10 minutes at 4,000 U / min and 10°C. 250 pL supernatant mixed with 250 pL acetonitrile and 500 pL 90% acetonitril:10% DI water solution. The mixture was vortexed for 30 seconds again and filtered through a 0.2 pm wwPTFE syringe filter into a cap. Priorto analysis 100 pL of the final extract mixed up with 100 pL ISTD (internal standard) solution containing Soya Saponin Ba. For quantification an external calibration with internal standard will be prepared in a concentration range from 0.1 to 1 ,500 ng / mL for Saponin B in 50% acetonitrile: 14% ethanol:26% DI water solution. Further dilution used: 8x. Samples and calibration were measured by a LC-(ESI)-MS / MS (Agilent HPLC 1290 Inf II with Agilent 6470 triple quad detector) method using a Agilent Poroshell 120HILIC-Z; 2,1x100mm 1 ,9 pm column with a DI water (+0,1 % formic acid) and acetonitrile (+0,1 % formic acid) gradient. MRM transition used: Soya Saponin Ba (ISTD): 960->441 m / z; Saponin B: 944 -> 441 m / z; Saponin DDMP: 1070 -> 423. The quantification of Saponin DDMP based on calibration with Saponin B as Saponin B equivalents due to lack of reference material. Results were calculated on dry matter (DM) content or fresh matter (FM) content. As it is shown in Table 4 below for selected comparative measurements made, KWS076 I KM 17AE076 indeed has a very favourable saponin profile, as both saponin B and saponin DDMP are significantly reduced in comparison to reference plants (here: Flam as reference; S in Table 4 used for “Saponin”) as shown for the AV (average) saponin values measured below as well as for the sum of saponins (B + DDMP; sum of average).

[0208] TABLE 4: Saponin content comparison

[0209] Therefore, it could again be confirmed that KWS076 is a highly interesting line for saponin and thus off- flavour reduced pea line development. Example 6: Deeper evaluation of candidates and establishment of SNP markers for signature mutations

[0210] Individual candidates influencing the saponin pathway in pea were then deeper analyzed in the relevant germplasm of interest and in various comparative lines.

[0211] Strikingly, many Pisum sativum candidate genes shown to be involved in the saponin biosynthesis pathway turned out to have only a low level of identity to homologs / orthologs / paralogs in other cultivars. This is exemplary shown for Pisum sativum TSAR genes in Table 5 showing a very low degree of sequence identity / conservation to Chenopodium quinoa genes when directly aligned.

[0212] TABLE 5: Exemplary sequence comparison

[0213] From this it was followed that signature mutations of interest had to be identified and evaluated on a case by case basis for pea, as it was not meaningful and successful to straightly extrapolate findings from other cultivars.

[0214] Example 7: Development and application of genetic fingerprinting marker sets for establishment of identity

[0215] To screen for signature mutations and to select relevant plant material, fingerprint markers were developed. This development consisted of analyses in whole genome sequencing data from the lines of interest. The specificity of these markers lies in the careful selection of variants where allele frequencies were very low, out of a given sample size these alleles were observed at frequencies lower or equal to 0.01 , in other words almost exclusively presently in said lines. This proves an effective way of establishing identity as this accounts for the combined probabilities of the observed markers at their respective allele frequencies.

[0216] The application of the marker set to establish identity consists of sequence analyses in the genomic regions, through whichever form of technology, along each of the different physical positions in the genome, 12 different physical positions for line 17AE076 deposited under NCIMB No. 44401.

[0217] In order for identity of each line to be established, for every given sequence position with a variant in question of the reported marker set, the allele observed must match that of the denoted SNP assigned to the respective line, only queries with 100% of matching genotype calls at each of the given marker positions are established with an identity of 17AE076. A detailed explanation is illustrated below in Table 6, where cells in light grey correspond to matching genotype calls and thus characterizing SNP signature marker positions between a queried individual and the corresponding genotype calls observed for line 17AE076 deposited under NCIMB No. 44401. Dark grey cells (and signature SNP position nucleotides marked in bold and underlined) denote mismatches in genotype calls thus resulting in a loss of identity.

[0218] FP01 to FP12 in Table 6 corresponds to SEQ ID NO: 10 to 21 , respectively.

[0219] When now wishing to enter the production of food ingredients using a germplasm with the favourable characteristics and signature mutations of the present invention for downstream (product I food) production, quality checks can be performed as follows: Usually, one will start with a high quality reference line having a phenotype to be improved (or a customer wishes to have improved). The material will then be included in routine in-house research as a frame of reference for improvement.

[0220] For this, usually three samples of genetic stocks will be submitted to include them in cross schemes for future generations. After analyzing the stocks in question, through genomics similarities between the lines as disclosed herein can be identified and a deeper screening through fingerprint marker sets, e.g. as shown in Queries 1-3 in Table 6 can follow.

[0221] In the example shown in Table 6, through direct comparisons of observed vs. expected genotype calls it was observed that one sample, Query 1 , has a 100% genotype call match to NCIMB No. 44401 line, i.e. 12 / 12 marker match.

[0222] For smart and quick screens, it will be enough to have less markers, but usually at least two markers, for unambiguously identifying a non-match.

[0223] This approach allows for direct identification of the genetics underlying NCIMB No. 44401 material and material derived therefrom in combination with a functional test for an improved taste profile following from the NCIMB No. 44401 genetics.

[0224] TABLE 6: SNP signature mutations within NCIMB No. 44401 17AE076_FP_11

[0225] Identity 17AE076 NCIMB No. 44401 17AE076 Unestablished Unestablished

[0226] Next, further candidates were screened that directly and indirectly influence the saponin biosynthesis pathway. Pisum sativum TSAR 1 to 3 (SEQ ID NO: 1 to 9) Pisum sativum alpha-1 ,2 mannosyltransferase (SEQ ID NO: 22 and 23), two variants of Pisum sativum cytochrome P450 72A68- like (SEQ ID NO: 24 to 27), Pisum sativum Terpene cyclase (SEQ ID NO: 28 and 29), and Pisum sativum specific beta-amyrin synthais - e (BAS, SEQ ID NO: 30 and 31) were evaluated in the relevant 17AE076 material deposited as NCIMB No. 44401 , as mutations were identified having an influence on a phenotypic level on the taste profile of the plant.

[0227] Notably, certain signature SNP mutations as shown in Table 7 below could be identified and markers were designed and tested (SEQ ID NO: 32 to 38) that, alone or in combination, allow the identification of material with a favourable signature mutation. i -

[0228] TABLE 7: Further SNP signature mutations within NCIMB No. 44401

[0229] The above results show that based on the analysis of the genome of 17AE076 deposited as NCIMB No. 44401 relevant genotypic and phenotypic findings could be made that are applicable for the specific line, but also beyond: generally applicable SNP signature mutations and genotype positions could be identified that are relevant for identifying and characterising pea plants having an improved taste profile useful for a variety of food applications, wherein it could be shown that signature mutations in at least one gene or a non-coding region thereof directly or indirectly involved in the saponin biosynthesis pathway are highly characteristic to rapidly and efficiently identify pea plants for producing plant-based ingredients with an attractive and improved flavour versus plants that do not carry the relevant mutations.

Claims

CLAIMS1 . A plant-based ingredient obtained from or obtainable from a pea of a Pisum sativum, wherein a representative sample of seeds of said plant was deposited under NCIMB No. 44401 , wherein the plant-based ingredient provides an improved taste profile as compared to a plant-based ingredient from a pea from a reference Pisum sativum plant, wherein the improved taste profile is due to at least one signature mutation in at least one gene, preferably a non-coding region of the gene, wherein the gene is involved in the saponin biosynthesis pathway.

2. The plant-based ingredient of claim 1 , wherein the pea plant comprises at least one signature mutation(s) of at least one nucleotide sequence of a gene preferably a non-coding region of the gene, affecting saponin biosynthesis pathway in the plant in comparison to a reference Pisum sativum plant, wherein the gene encodes and is selected from a cytochrome P450 monooxygenase (P450), a uridine diphosphate-dependent (UDP) glycosyltansferase (UGT), a TSAR transcription factor, a basic leucine zipper (bZIP) transcription factor, or a squalene synthase, including a cytochrome P450 monooxygenase (P450), a uridine diphosphatedependent glycosyltansferase (UGT), and / or a Triterpene Saponin Biosynthesis Activating Regulator (TSAR) transcription factor, including TSAR1 , wherein the mutation reduces, modifies or eliminates the biosynthesis of saponin in the plant or a plant part in which it is expressed, optionally wherein the plant comprises at least one further mutation affecting the taste profile in a gene involved in a pathway other than the saponin pathway, preferably in a lipoxygenase biosynthesis pathway, or wherein at least one signature mutation is identified by at least two markers, wherein said markers are individually selected from a combination of markers of SEQ ID NO: 10 to 21 , or a modified marker thereof provided that the modified marker still comprises or encodes the relevant SNP signature mutation of a plant deposited under NCIMB No. 44401 at the relevant genotype position as shown in Table 6 and / orwherein at least one signature mutation is identified by at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or eight markers, wherein said markers are individually selected from a combination of markers of SEQ ID NO: 32 to 38, or a modified marker thereof provided that the modified marker still comprises or encodes the relevant SNP signature mutation as shown in Table 7.

3. The plant-based ingredient according to claim 1 or 2, wherein the improved taste profile is independently selected from the group consisting of an improved flavour profile, including a less bitter flavour, a less sour flavour, a less beany flavour and / or a less pea-like flavour, an improved mouthfeel, including a less powdery mouthfeel, a less dull mouthfeel and / or a less coating mouthfeel, a less bitter aftertaste, an improved aroma, including as a less pea-like aroma and / or a less green / grassy aroma, or any combination thereof.

4. The plant-based ingredient of any of the preceding claims, wherein the ingredient is a protein, a starch, a fat, or combination thereof, preferably a protein.

5. A plant-based composition comprising a plant-based ingredient of any one of the preceding claims.

6. The plant-based composition of claim 5, further comprising:(i) a second plant-based ingredient from a plant different from the first plant-based ingredient; and / or(ii) a protein ingredient obtained from an animal source, from a microbe, a fungi, fermentation and / or cell culture to obtain a hybrid composition.

7. The plant-based composition of claim 5 or 6, wherein the composition is a protein composition.

8. An alternative consumable product comprising a plant-based ingredient or a plant-based composition according to any one of claims 1 to 7.

9. The alternative consumable product of claim 8, wherein the alternative product is selected from an alternative beverage product, including an alternative dairy beverage, an alternative non-dairy beverage, or an alternative fermented beverage, or wherein the alternative product is selected from an alternative consumable product, including an alternative dairy product, an alternative seafood product, an alternative egg product and an alternative meat product.

10. The alternative consumable product of claim 8 or 9, wherein the product is a vegetarian or a vegan product.

11. A method of producing an alternative consumable product of any one of claims 8 to 10, comprising (i) providing a plant-based ingredient or a plant-based composition of any one of claims 1 to 7; ii) adding at least one further additive and / or ingredient; (iii) producing an alternative consumable product.

12. A use of a plant-based ingredient or composition according to any one of claims 1 to 7 for reducing or eliminating off-flavours in an alternative consumable product, preferably in an alternative consumable product as defined in any of claims 8 to 10.

13. A method of producing a plant-based ingredient of claims 1 to 4, comprising the steps of:(i) providing a seed from the Pisum sativum plant having a representative sample of seeds deposited under NCIMB No. 44401 , or optionally of a progeny thereof; and(ii) extracting a fraction, including a protein fraction, a starch fraction or a fat fraction, from the pea, preferably wherein the extracting step is a dry extraction method or a wet extraction method.

14. The method of claim 13, wherein the method produces a protein ingredient in the form of a concentrate, an isolate, a texturate, a powder, a flake, or a flour.

15. A use of a Pisum sativum plant or seed for producing a plant-based ingredient according to any one of claims 1 to 4, wherein said plant or seed is or is a progeny of a seed deposited underNCIMB No. 44401 , preferably wherein the at least one Pisum sativum plant or seed is characterized by at least two markers, wherein each marker identifies a SNP signature mutation, wherein said markers are individually selected from a combination of markers of SEQ ID NO: 10 to 21 , or a modified marker thereof provided that the modified marker still comprises or encodes the relevant SNP signature mutation of said plant deposited under NCIMB No. 44401 as shown in Table 6 and / or wherein the at least one Pisum sativum plant or seed is characterized by at least one signature mutation identified by at least one, at least two, or more marker(s), wherein said marker(s) is / are individually selected from a combination of markers of SEQ ID NO: 32 to 38, or a modified marker thereof provided that the modified marker still comprises or encodes the relevant SNP signature mutation as shown in Table 7, preferably wherein a BAS specific marker according to SEQ ID NO: 32, or a modified marker thereof, is used in combination with at least one, at least two, or more markers) of SEQ ID NO: 10 to 21 , or a modified marker thereof. .

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