Pea varieties with an improved flavor profile

Abstract

A plant-based ingredient obtained from or derived from a Pisum sativum pea, wherein a representative sample of seeds of this plant has been deposited under NCIMB No. 44401, wherein the plant-based ingredient has an improved taste profile compared to a plant-based ingredient derived from a pea of ​​a Pisum sativum reference 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.

Description

AREA OF INVENTION

[0001] This disclosure relates to plant-based ingredients, in particular protein ingredients, suitable for use in foods, including vegetarian or vegan meat or dairy analogues. The plant-based ingredients are obtained from a novel line of peas, which imparts an improved flavor profile to their ingredients. BACKGROUND OF THE INVENTION

[0002] The challenges arising from continuous population growth, malnutrition and obesity, as well as environmental changes, have led to strong market interest in plant-based ingredients in recent years. Food manufacturers face the challenge of providing plant-based food alternatives on a large scale that meet consumer expectations in terms of taste, texture, and other organoleptic properties.

[0003] To meet these requirements, food manufacturers have considered various plant-based protein sources, including but not limited to soybeans, peas, chickpeas and split peas, lupins, hemp, potatoes, wheat, cranberries, beans such as marine, pinto, adzuki, fava, lima, black, red kidney and mung beans, pumpkin seeds and seeds of other squashes, and grains such as rice, sorghum and millet.

[0004] One of the main limitations of using plant-based ingredients, such as plant-based proteins, in food products is the perception of off-flavors. These off-flavors can be mitigated to some extent by natural flavorings and flavor-masking agents. However, the presence of certain aroma- and flavor-active compounds in plant-based isolates or concentrates makes the product development process cumbersome, costly, and time-consuming, and may also be perceived as undesirable by consumers.

[0005] Peas, as a plant-based ingredient source, particularly as a protein source, have attracted considerable interest from the food industry and consumers in recent years. The endogenous nitrogen source in peas reduces the need for soil nitrogen and is responsible for their high protein content, making peas an excellent source of plant-based protein. Furthermore, as a high-quality protein source, peas have a much lower carbon footprint (CO2 in g / 100 g of protein produced) compared to meat-based protein sources or eggs (from chickens) (Sustainable Protein Sources. 1st edition, 2016, Editors: Sudarshan Nadathur, Janitha PD Wanasundara, Laurie Scanlin, eBook ISBN: 9780128027769).

[0006] However, it has been shown that cultivated varieties of Pisum sativum L. differ considerably 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.

[0007] Peas, as a plant-based ingredient source, particularly as a protein source, have therefore attracted considerable interest from the food industry and consumers in recent years. The endogenous nitrogen source in peas reduces the need for nitrogen in the soil and is responsible for their high protein content, making peas an excellent source of plant-based protein. Furthermore, pea protein is associated with better digestibility and relatively fewer allergenic reactions and negative health controversies compared to soybeans or other plant-based proteins.

[0008] Furthermore, it has been shown that cultivated varieties of Pisum sativum L. differ significantly 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).

[0009] These differences in chemical composition necessitate specialized manufacturing methods to develop the food products desired by consumers, particularly novel alternative food products, and parameters that may require individual batch-to-batch adjustments or continuous process modifications, thus limiting the possibilities for manufacturing or using pea ingredients on a large scale. Such differences also lead to variations in the properties of the ingredients and the final product, including organoleptic properties such as taste or aroma, making the use of pea ingredients in commercial food production challenging.

[0010] A non-volatile compound found in peas and other plants is saponin, or more precisely, the group of saponin compounds. Saponins, including group A saponins (also referred to here as saponin A), group B saponins (also referred to here as saponin B), 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP) saponins, and group E saponins (also referred to here as saponin E), are amphiphilic glycosides of steroids and triterpenes and are known to cause bitter, beany, grassy, ​​and astringent flavors, which limits the use of saponin-containing plant-based ingredients in various foods and other consumer products.To overcome these limitations, it would be advantageous to enable the production of plant-based ingredients such as proteins, including pea proteins, which have a lower or modified saponin content in order to reduce, significantly reduce, or eliminate the off-flavors and odors typically associated with such plant-based ingredients.

[0011] However, plant saponin content fulfills an important defense function in the plant. Therefore, completely knocking out key genes of the saponin pathway, leading to the elimination or significant reduction of total saponin content, can in some cases result in reduced plant vitality, non-viable plants, or reduced crop yields. Consequently, this could necessitate the use of externally applied fungicides and other treatments to overcome this deficiency. In the food sector, the use of fungicides and other treatments is generally discouraged by food and feed manufacturers as well as end consumers.Therefore, a balance is needed between adapting plant-based ingredients for the desired organoleptic properties and providing solutions that deliver a sufficient yield of plants and plant materials to be sustainable.

[0012] There is therefore a constant need for pea constituents that exhibit fewer off-flavors, improved taste and organoleptic properties. Furthermore, there is a great need to define and develop suitable germplasm and pea lines that can be cultivated sustainably and contain plant-based protein constituents with high edibility, particularly nutritional value. BRIEF DESCRIPTION OF THE IMAGES

[0013] Fig. ( Fig. Figure 1) shows a differential plot of Kameleon peas and KWS076 (synonym: KM 17AE076, deposited under the Budapest Treaty as NCIMB 44401) peas for comparison, as described in Example 3 below. AR means aroma, FL means taste, AT means aftertaste.

[0014] Fig. ( Fig. Figure 2) shows a differential diagram of an alternative milk drink, as described in more detail in Example 4 below. AR means aroma, FL means taste, AT means aftertaste. BRIEF DESCRIPTION OF THE SEQUENCES SEQ ID NO Beschreibung 1 TSAR1_ZW6_WGS regulatorisch 2 TSAR2_ZW6_WGS regulatorisch 3 TSAR3_ZW6_WGS regulatorisch 4 TSAR1_CDS 5 TSAR2_CDS 6 TSAR3_CDS 7 TSAR1 Protein 8 TSAR2 protein vorausgesagt 9 TSAR3 protein vorausgesagt 10 17AE076 FP 01 11 17AE076 FP 02 12 17AE076 FP 03 13 17AE076 FP 04 14 17AE076 FP 05 15 17AE076 FP 06 16 17AE076 FP 07 17 17AE076 FP 08 18 17AE076 FP 09 19 17AE076_FP_10 20 17AE076_FP_11 21 17AE076_FP_12 22 CDS_Pisum sativum alpha-1,2-Mannosyltransferase 23 Protein_Pisum sativum alpha-1,2-Mannosyltransferase 24 CDS_Pisum sativum Cytochrom_P450 72A68- like 1 25 Protein_Pisum sativum Cytochrom P450 72A68- like 1 26 CDS_ Pisum sativum Cytochrom P450 72A68- like 2 27 Protein_ Pisum sativum Cytochrom P450 72A68- like 2 28 CDS_ Pisum sativum Terpen-Cyclase 29 Protein_ Pisum sativum Terpen-Cyclase 30 CDS_ Pisum sativum beta-Amyrin-Synthase 31 Protein_ Pisum sativum beta-Amyrin-Synthase 32 17AE076_BAS 1 Markierung 33 17AE076_Cytochrom-Markierung 1 34 17AE076_Cytochrom Markierung 2 35 17AE076_ Cytochrom-Marker 3 36 17AE076_ Cytochrom-Marker 4 37 17AE076_Cytochrom-Markierung 5 38 17AE076_Cytochrome label 6 DEFINITIONS

[0015] As used herein, “a”, “an”, or “the” can refer to one or more than one. For example, “a” cell can mean a single cell or a multitude of cells. Furthermore, the term “a plant” can encompass a multitude of plants.

[0016] A “dry extraction process” comprises protein extraction processes in which the protein fraction is obtained from the starting material using air, pressure, or other non-hydrating methods, or combinations thereof. A “wet extraction process” comprises protein extraction processes in which the protein fraction is obtained from the starting 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.

[0017] The term "aroma", as used herein, refers to a property that can be detected by taste and / or smell.

[0018] The term "non-volatile flavor components," as used herein, refers to molecules that impart a flavor to a product but are not volatile in the sense that they cannot be removed by methods such as evaporation. The term applies to both liquids and solids.

[0019] The term “off-flavor” or “off-flavors,” as used herein, refers to an undesirable flavor and encompasses the complex interplay of various taste descriptors such as bitter, sour, earthy, beany, salty, sweet, green, nutty, umami, and the like. Furthermore, other organoleptic parameters such as dry, dull, powdery, moist, and the like also play a role in taste perception and are collectively referred to here as “off-flavors.” The term “plant-based ingredient,” as used herein, refers to any ingredient obtained from plants or plant parts in 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 plant-based ingredient may be any combination of proteins, carbohydrates, and fats or ash, etc.contained in any proportion. The term "plant-based ingredient" may be used interchangeably with the term "plant-based food ingredient" herein. The terms "protein ingredient," "starch ingredient," "carbohydrate ingredient," "lipid ingredient," or "ash ingredient," as used herein, include any form of such ingredient suitable for use on its own or in combination with other components of a formulation, such as the formulation for a plant-based composition or an (alternative) food product.

[0020] The plant-based ingredient can be in the form of an isolate, concentrate, flour, textured product, and the like.

[0021] “Protein textureate” or “textured protein,” as used herein, refers to an ingredient that possesses structural integrity and an identifiable structure such that individual units, occurring as fibers, slivers, chunks, pieces, granules, discs, and the like, can withstand hydration and cooking or other processes of preparing food for consumption. In general, textured proteins can be used to modify or enhance texture and to bind water. Edible protein sources from which textured proteins are produced include, but are not limited to, pulses (e.g., pulse proteins), peas, soybeans, corn, wheat, chickpeas, potatoes, rice, sunflowers, and the like.Textured proteins 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 texturing proteins are known and described in the scientific community and may include, for example, high-temperature and pressure extrusion, spinning, freeze texturing, chemical or enzymatic texturing, and the like.

[0022] “Protein flour”, as used herein, refers to an ingredient containing ground peas.

[0023] An “alternative edible product ingredient,” as used herein, refers to a plant-based ingredient, including a protein ingredient, that is suitable as part of an “edible product” or “edible composition.” Furthermore, at least a part or fraction of the “alternative edible product ingredient” is suitable as a substitute for a well-known and commonly manufactured product ingredient, typically being suitable as an alternative to, for example, animal-derived or animal-produced ingredients or foods, including meat, eggs, or dairy products.

[0024] An “alternative food,” as used herein, may be in liquid, semi-liquid, or solid form. An “alternative food,” as used herein, refers to a product for human or animal consumption that is normally made with ingredients from animal sources, but in which the animal ingredient has been partially or completely replaced by a plant-based substitute ingredient. Non-restrictive examples of alternative food products include alternative beverages such as a milk substitute or a drinkable yogurt substitute, or alternative food products such as a substitute for eggs, beef, dairy products, poultry, or seafood. Alternative foods also include pet or animal feed products in which some or all of the animal-derived ingredients are replaced by plant-based ingredients.The exchange or replacement of the animal-derived component may, in some embodiments, amount to more than 70%, 80%, 90%, or 95%. In other embodiments, the exchange or replacement of ingredients of animal origin may amount to 70% or less, e.g., 60%, 50%, 40%, etc. Alternative foods may also include dairy-free beverages such as sports drinks or smoothies. Alternative foods may also refer to alternative food products, such as plant-based powders used as dietary or nutritional supplements.

[0025] An “alternative food,” “alternative diet,” or “alternative (food / cosmetic) product / composition,” as used herein, refers to a food, including liquid foods such as beverages, that is usually plant- or microorganism-based and provides an alternative to foods of animal origin, including meat or dairy products. Alternative foods may be of particular interest as alternative protein sources, but the term “alternative food” refers to any type of nutrient building block, including proteins, carbohydrates, lipids, vitamins, minerals, fiber, and the like, that is suitable for food production and that is well-accepted or even healthy as food and feed for humans or farm and domestic animals.An alternative food product or foodstuff thus represents an "alternative edible product" or an ingredient thereof. The ingredients of the present invention are suitable for the production of an alternative foodstuff, an alternative feed, or an alternative cosmetic.

[0026] An alternative product or composition, as used herein, and any pea protein ingredient suitable for the manufacture 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 is typically processed, isolated, concentrated, and / or otherwise treated to be incorporated into an alternative product or composition. Furthermore, a pea protein ingredient of this invention typically represents an intermediate ingredient that has been or can be isolated from a plant that constitutes a part or fraction of a final product or composition, or a mixture or hybrid product.In an alternative product or composition, the pea protein ingredient of this invention, as an alternative part or fraction of the alternative product or composition, thus replaces a part or fraction that would be present in a generally known and commonly produced product or composition, preferably replacing a part or fraction of animal or non-plant origin in the corresponding generally known and commonly produced product.

[0027] In particular, the term ‘food’ as used herein refers to food intended for human consumption, while ‘feed’ as used herein refers to animal feed.

[0028] “Milk substitute” or “milk substitute composition” or “milk alternative” or “milk alternative product”, as used herein, refers to compositions that mimic the general appearance, nutritional content, and / or taste of dairy products made using animal milk products without containing animal-based milk or being substantially free of animal products, and includes hybrid products made with laboratory-grown, fermented, and animal-derived components, such as protein components. The milk substitute may be completely free of animal milk or animal milk protein, or virtually free of animal milk protein, such as 90% or 95% free of animal milk protein. The milk substitute may be a dairy-free cheese, a dairy-free yogurt, a dairy-free ice cream, and the like.

[0029] “Meat substitute” or “meat substitute composition” or “meat alternative”, as used herein, refers to compositions that mimic the general, organoleptic, and / or nutritional properties of edible products manufactured using any type of meat or meat analogue, including meat, fish, poultry, laboratory-grown, and fermented meat products. This definition also includes hybrid products made from laboratory-grown, fermented, and animal 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” are used herein, they refer to a composition of any of the protein constituents, flours, concentrates, or isolates disclosed herein, used in extracted form together with other ingredients of different origins to provide said composition. An alternative food or cosmetic product is thus also a composition in this sense. In this context, a “mixture” or “pea protein mixture” is a specific type of composition in which pea protein fractions from different peas, or even from different plants or other sources, are blended together to provide a base protein mixture (and optionally other additives or ingredients of different types and / or origins) containing pea protein of the present invention.

[0030] A “consumer,” as used herein, refers to goods that are typically consumed or used up in the normal course of business, such as food and beverages, office supplies, cleaning and sanitation products, and medical products. There are generally two main types of consumables: durable consumables, which can be used over a long period, and non-durable consumables, which can be used up relatively quickly. Examples of consumables include perishable food and beverages, paper products, ink cartridges, cleaning chemicals, gloves, and syringes.The consumable products specifically addressed here are non-permanent products that are non-toxic when swallowed or applied to the human or animal body, according to their intended use, and are typically manufactured from at least one organic raw material (and optionally others), including foods, beverages, gels, ointments, toothpaste, and the like. "Consumable products / compositions" within the meaning of the present invention represent alternative products or compositions comprising at least one alternative plant-based ingredient, preferably a protein ingredient, as defined in the present invention.

[0031] A “gene,” as used herein, refers to the coding region of a gene, the non-coding region, and upstream and / or downstream regulatory sequences, including enhancers, silencers, promoter elements (e.g., proximal, distal, and nuclear promoters), and 5' and / or 3' UTRs. Therefore, the modification of a gene may also include the modification of a non-coding and / or regulatory sequence thereof.

[0032] A “hybrid composition / product” or a “hybrid alternative (consumer, including food / cosmetic, etc.) composition / product” as used herein refers to a product that at least partially comprises an “alternative food” or “alternative diet” or an “alternative (food) product” that includes a protein substance of plant origin according to the present invention, but which may include further components.

[0033] A "knockdown" of a gene refers to an experimental technique in which the expression of the gene (i.e., the transcription of DNA into RNA and thus the amount of active RNA transcripts) is reduced. Reduced expression can be achieved, for example, by gene silencing, in which the transcription rate is reduced or stopped, thereby decreasing the amount of available functional RNA.

[0034] A "knockout," on the other hand, leads to a complete loss of expression (transcription / translation). This means the gene is not fully transcribed or is transcribed incorrectly, resulting in either complete loss of expression or (at the protein level) a reduction in expression to such an extent that almost no functional protein is expressed, or expression is very low, at or above the detection limit. This can be achieved, for example, by exchanging or interrupting the sequence of the target gene. For instance, an early or premature additional stop codon, preferably near the start codon, can lead to premature transcription termination, resulting in the complete loss of the functionally translated protein. Alternatively, a knockout can be achieved by a mutation that alters the naturally occurring splice donor or splice acceptor site of a eukaryotic gene, encompassing exons and introns.The splice donor site typically comprises a nearly invariant GU sequence 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 a nearly invariant AG sequence. If these conserved sequences are mutated, RNA splicing is altered, which can also lead to a knockout, measured at the transcriptional (RNA) or translational (protein) level. Consequently, a single targeted nucleotide exchange, deletion, or insertion can result in a functional knockout in the sense that the sequence encoded by a gene is no longer transcribed or translated. A knockout can also occur through a deletion of a gene or a substantial portion thereof at the genomic level, possibly accompanied by substitution with another sequence.

[0035] A “mutation” or “genome modification” within the scope of the present invention refers to any modification of a coherent nucleic acid sequence by modifying a nucleic acid sequence at a specific nucleotide sequence position, resulting in at least one difference in the (nucleic acid) sequence that distinguishes it from the original sequence. A modification can be achieved, in particular, by the insertion or addition of one or more nucleotides, or by the substitution or deletion of one or more nucleotides of the original sequence, or by any combination thereof.

[0036] A “nucleic acid construct”, “construct”, or “expression construct” refers to a nucleic acid molecule that encodes or comprises 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), polypeptide(s), or protein(s). A nucleic acid construct may also include regulatory sequences such as promoter and terminator sequences that facilitate the expression of the genetic element(s), as well as spacers and introns. The genetic elements of the present invention may also be encoded on a set of constructs, the constructs of which can be introduced into a cell simultaneously or sequentially.

[0037] The term “RNAi,” or “RNA silencing,” or “gene silencing,” as used interchangeably herein, refers to the process known as RNA interference, i.e., a mechanism for downregulating (or knocking down) genes, which has now been demonstrated in all eukaryotes. The mechanism was originally identified and described in plants, where it was termed “post-transcriptional gene silencing,” or “PTGS.” In RNAi, small RNAs serve to direct specific effector proteins to a target nucleotide sequence through complementary base pairing, resulting in the degradation of the target. A “gene silencing construct” or “RNAi agent” typically includes so-called “sense” and “antisense” sequences. Sense and antisense sequences are complementary sequences that exist in reverse orientation within 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 during transcription, resulting in an "RNA hairpin". In an RNA hairpin, sense sequences and their corresponding antisense sequences together form a double strand and are separated by an "interposed intron loop sequence" that forms the loop of the hairpin structure.

[0038] The terms Pisum sativum (L.) plant and pea plant, or simply pea, are used interchangeably here, whereby the term pea is used in connection with the plant as a whole, but also to refer to parts of it, especially seeds / fruits in pea pods.

[0039] A “(pea) plant constituent,” as used herein, refers to the total amount of protein that can be extracted from a (pea) plant fruit or seed (dry or fresh). A “(pea) protein constituent,” in turn, refers to the total amount of protein in the (pea) plant.

[0040] The term "protein concentrate" refers to a protein ingredient with a concentration of approximately 30% to 60%. A "protein isolate" is an even more concentrated protein ingredient with a concentration of approximately 60% to approximately 100%. "Protein flour" represents the protein that can be obtained directly after dehulling and milling. Because this protein flour is not yet heavily processed, it reflects the original protein content quite directly. Therefore, "protein flour" was also used by the inventors to define standard ratios when comparing different materials. "Protein textured" or "textured vegetable protein" refers to a flour product that has usually been further defatted and is particularly well-suited and used as a meat analogue or meat extender. It cooks quickly and has 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 typically composed of fine, dry particles produced by grinding, crushing, or dissolving a solid substance.

[0041] The term “vector” refers to an element used to introduce a nucleic acid construct or set of nucleic acid constructs into a cellular system. The vector may be a plasmid or plasmid vector, a cosmid, yeast artificial chromosomes (YAC), a bacterial artificial chromosome (BAC) or P1 artificial chromosomes (PAC), a phagemid, a 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 as disclosed herein.

[0042] The terms "plant," "plant cell," or "plant part," as used herein, refer to a plant organism, a plant organ, differentiated and undifferentiated plant tissues, plant cells, seeds, and their derivatives and progeny. Plant cells include, without limitation, cells from seeds, from mature and immature cells or organs, including embryos, meristematic tissue, seedlings, callus tissue at various stages of differentiation, leaves, flowers, roots, shoots, male or female gametophytes, sporophytes, pollen, pollen tubes and microspores, as well as protoplasts, etc.

[0043] "Mutagenesis" refers to a technique in which changes or mutations are introduced into a nucleic acid sequence in a random or non-site-specific manner. Mutations can be induced, for example, by certain chemicals such as EMS (ethyl methanesulfonate) or ENU (N-ethyl-N-nitrosourea), or physically, such as 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 specific target sequence and allow a modification to be made at a specific location within that sequence.

[0044] A “protein composition,” as used herein, refers to a protein isolate that can be obtained directly from a fruit, a seed, in particular from a specific pea of ​​the genus Pisum, or from a flour, protein fraction, or purified or partially purified protein fraction. Depending on the method of its preparation, the protein composition may contain denatured and / or partially fragmented proteins, as the proteins may have been denatured and / or fragmented during thermal, chemical, and / or mechanical processing / purification. A protein composition may consist substantially of one protein or a fragment thereof, in particular convicilin, or it may be a mixture of protein compositions containing other proteins, in particular globulins and other pea proteins.

[0045] TILLING (Targeting Induced Local Lesions in Genomes) is a method used to identify mutations in a specific gene after (non-specific) mutagenesis. The mutagenesis can be performed, for example, with a chemical mutagen such as EMS. Subsequently, a sensitive DNA screening method is used to identify mutations at individual bases. Methods for performing TILLING are known to those skilled in the art.

[0046] A “functional homolog,” as used herein, refers to a molecule that has essentially the same function as a reference molecule. A “structural homolog” refers to a homolog that has a substantial degree of sequence identity with a reference molecule or a part thereof from which it is derived.

[0047] The term "nucleotide sequence" in connection with the present invention includes genomic DNA, cDNA, synthetic DNA and RNA. Preferably, it refers to DNA, in particular genomic DNA.

[0048] Amino acids are referred to here by their name, three-letter abbreviation, or one-letter abbreviation. The term "protein," as used herein, includes proteins, polypeptides, and peptides. The term "amino acid sequence," as used herein, is synonymous with "polypeptide" and / or "protein." In some cases, "amino acid sequence" is synonymous with "peptide." In some cases, "amino acid sequence" is synonymous with "enzyme."

[0049] A "conservative amino acid substitution" is a substitution in which the amino acid residue is replaced by an amino acid residue with a similar side chain. In the scientific community, families of amino acid residues with similar side chains have been defined. 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), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, a predicted non-essential amino acid residue in a protein according to the invention is preferably replaced by another amino acid residue from the same side chain family.

[0050] Conservative amino acid substitutions can occur along the entire length of the polypeptide sequence of a functional protein, such as an enzyme. In one embodiment, such mutations do not affect the functional domains of an enzyme. In another embodiment, the conservative mutations do not affect the catalytic sites of an enzyme.

[0051] In the present disclosure and the claims, the conventional one- and three-letter codes for amino acid residues can be used. The three-letter code for amino acids is defined in accordance with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). Due to the redundancy of the genetic code, a polypeptide can also be encoded by more than one nucleotide sequence.

[0052] A plant line that is “homologous” or a “homologous line” to KWS076, as used herein, refers to a line created by modifying or introducing at least one gene locus of KWS076 based on the present disclosure to selectively modify a gene in the saponin pathway in accordance with the present disclosure based on the knowledge and teachings provided for KWS076. Furthermore, orthologous genes to those identified herein may also be derived and used to provide plants with an optimized saponin biosynthesis pathway, e.g., in other plants of the family Fabaceae, formerly Leguminosae (here used interchangeably).

[0053] Where this disclosure refers to the percentage of identity between nucleic acid or amino acid sequences, or to the identity of a homolog, ortholog, or parolon of a particular gene locus with another, these values ​​define the values ​​obtained using the EMBOSS Water Pairwise Sequence Alignments (nucleotide) program (www.ebi.ac.uk / Tools / psa / emboss_water / ) for nucleic acids or the EM-BOSS Water Pairwise Sequence Alignments (protein) program (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 entire length of two sequences being compared. The tools provided by the European Molecular Biology Laboratory (EMBL) and the European Bioinformatics Institute (EBI) for local sequence matching use a modified Smith-Waterman algorithm (see www.ebi.ac.uk / Tools / psa / and Smith, TF & Waterman, M.).See “Identification of common molecular subsequence” Journal of Molecular Biology, 1981 147 (1):195-197). When performing an alignment, the standard parameters defined by EMBL-EBI are used. These 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. It is known to those skilled in the art that, for example, a sequence encoding a protein can be “codon-optimized” if the sequence in question is to be used in a different organism than the one from which the molecule originally came.

[0054] “Protein percent” or “starch percent” is usually measured by NIR spectrophotometry, but can also be measured using other methods known in the scientific community.

[0055] The term "plant" as used herein includes whole plants, including their offspring or descendants. As used herein, unless explicitly stated otherwise, the term "plant" refers to a plant at any stage of development. The term "plant part" includes any part or derivative of the plant, including certain plant tissues or structures, plant cells, plant protoplasts, plant cells or tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells intact in plants or plant parts, such as seeds, kernels, spadixes, flowers, cotyledons, leaves, stems, buds, roots, root tips, straw, and the like. Plant parts may also include processed plant parts or derivatives, such as flour, oils, extracts, protein fractions, etc. "Plant parts" include, for example, vegetative shoot organs / structures, e.g.,Leaves, stems, and tubers; roots, flowers, and floral organs / structures, e.g., bracts, sepals, petals, stamens, carpels, anthers, and ovules; seeds, including embryo, endosperm, and seed coat; fruits and mature ovaries; plant tissues, e.g., vascular tissue, soil tissue, and the like; and cells, e.g., sentinel cells, ovules, pollen, trichomes, and the like; as well as their offspring. Plant parts may be attached to or detached from a whole, intact plant. Such plant parts include, among others, organs, tissues, and cells of a plant, 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 exist as an isolated single cell or a cultured cell, or as part of a higher organized unit such as plant tissue, a plant organ, or a whole plant."Plant cell culture" means cultures of plant units such as 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 parts of flowers, fruits, pollen, ovules, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant. This includes callus or callus tissue, as well as extracts (such as taproot extracts) 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, refers to a group of plant cells that form a structural and functional unit. Any tissue of a plant, whether in planta or in culture, is included.This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue cultures, and all groups of plant cells organized into structural and / or functional units. The use of this term in conjunction with, or in the absence of, a particular type of plant tissue as listed above or otherwise covered by this definition is not intended to exclude 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 a seed, pollen, egg cell, protoplast, inflorescence, embryo, or callus.

[0056] As used herein, “control plant,” “control plant part,” “control cell,” or “control seed” refers to a plant, plant part, plant cell, or seed that is not subject to the procedures and compositions described herein. A “control,” “control plant,” “control plant part,” “control cell,” or “control seed” provides a reference point for measuring changes in the phenotype of the plant or plant cell in question. A control plant or cell may, for example, include: (a) a wild-type plant or cell, i.e., of the same genotype as the starting material for the genetic modification that resulted in the plant or cell in question; (b) a plant or plant cell of the same genotype as the starting material but transformed with a null construct (i.e.,with a construct that has no known effect on the trait of significance, such as a construct that includes a marker gene); c) a plant or plant cell that is an untransformed segregant among the offspring of a plant or plant cell concerned; d) a plant or plant cell that is genetically identical to the plant or plant cell concerned but is not exposed to any conditions or stimuli (e.g., sucrose) that would induce expression of the gene of significance; or e) the plant or plant cell concerned itself under conditions in which the gene of significance is not expressed. In certain cases, a control plant of the present disclosure is cultivated under the same environmental conditions (e.g., the same or similar temperature, humidity, air, soil, water, and / or pH conditions) as a plant concerned described herein.Similarly, a control protein or control protein composition may refer to a protein or protein composition isolated from or derived from a control plant. In certain 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 regulatory region of a gene, as disclosed in the present invention.

[0057] “Offspring” comprises an F1 pea plant resulting from the cross of two pea plants, wherein at least one plant comprises a pea line, variety or cultivar represented by a sample of seeds deposited or crossed with NCIMB 44401 in accordance with the terms of the Budapest Treaty, and offspring further includes, but is not limited to, subsequent F2, F3, F4, F5, F6, F7, F8, F9 and F10 generation crosses with the recurring parent line.

[0058] As used herein with respect to a parameter, the term "decreased," "decreasing," "decline," "reduced," "minor," 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 compared to a comparative control, such as a specified normal or reference value of the parameter, or a specified standard control. Accordingly, the terms "decreased," "reduced," and the like include a partial reduction compared to a control. As used herein, the terms "eliminate," "eliminated," or "eliminated" include a complete or near-complete reduction compared to a control.They refer, for example, to a 96%, 98% or 100% negative change in the parameter compared to a control.

[0059] As used herein, the terms “modification” or “modify” or other variations of the term, in the context of saponin content or the expression of saponin-related genes, refer to a modulation of the total saponin content by varying the saponin profile, as described in more detail below. For example, if saponin B content is reduced while DDMP saponin content is increased, the total saponin content does not change substantially; rather, the saponin profile is modified as a result of modifications to the expression of saponin-related genes.

[0060] As used here in relation to a parameter, the term "elevated," "increasing," or "increase" refers to a detectable (e.g., at least approximately 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 parameter compared to a comparative control, such as a specified normal or reference value of the parameter or a specified standard control. Accordingly, the terms "elevated," "increase," and the like encompass a slight, moderate, or significant increase compared to a control. DETAILED DESCRIPTION OF THE INVENTION

[0061] The inventors in this case have discovered, characterized and further developed a new line of peas that gives its ingredients and the alternative foods produced from them a particularly good taste profile.

[0062] In a first aspect, a plant-based ingredient, preferably a protein ingredient, is provided, which is obtained or available from a Pisum sativum pea, wherein a representative sample of seeds of this plant has been deposited under NCIMB No. 44401. The plant-based ingredient exhibits an improved flavor profile compared to a plant-based ingredient from a pea of ​​a Pisum sativum reference plant, wherein the improved flavor profile is attributable 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.

[0063] In one embodiment, the signature mutation is located in at least one gene involved in the saponin biosynthesis pathway, in a gene or its non-coding region that codes for a specific enzyme or protein directly involved in the saponin biosynthesis pathway.

[0064] In another embodiment, the signature mutation is part of the germplasm deposited under NCIMB No. 44401, wherein the mutation directly or indirectly affects the saponin biosynthesis pathway.

[0065] In one embodiment of the first aspect, the plant-based ingredient, preferably a protein ingredient, is obtained or available from a Pisum sativum pea, wherein a representative sample of seeds of this plant has been deposited under NCIMB No. 44401, carrying at least one signature mutation, can be identified 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 the 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 includes or encodes the relevant SNP signature mutation of one of the mutations deposited under NCIMB No. 44401 at the position marked with an ambiguity symbol in SEQ ID NO: 10 to 21, as shown in Table 6 for the mutation listed under NCIMB No.44401 deposited material shown and explained, i.e., where the relevant genotype position is at nucleic acid number 101 of SEQ ID NO: 10 to 21, and where the nucleic acid at the relevant genotype position is as follows: 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.

[0066] 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 the 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 includes or encodes the relevant SNP signature mutation (position indicated with an ambiguity symbol in SEQ ID NO: 32 to 38), as shown in Table 7, preferably using a BAS-specific marker according to SEQ ID NO: 32.

[0067] In particular, 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, enables both the accurate identification of signature mutations that are characteristic of NCIMB No. 44401 and simultaneously characteristic of the enhanced flavor profile of the relevant plant-based ingredient obtained or derived from the pea or 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.

[0068] Preferably, a plant-based ingredient according to the various aspects and embodiments of this disclosure contains no genetic information and the plant-based ingredient is not capable of self-replication or of being replicated in a biological system.

[0069] Since each of the markers of SEQ ID NO: 10 to 21 includes at least one characterizing signature mutation or single nucleotide polymorphism (SNP), as indicated by an IUPAC ambiguity symbol at the corresponding genotype position, as explained in the examples below, where the signature mutations are characteristic for NCIMB No. 44401, these signature mutations enable the accurate identification of the relevant plant germplasm, the plant germplasm resulting in a plant-based ingredient with an improved flavor profile compared to a plant-based ingredient from a pea from a Pisum sativum reference plant.

[0070] A “marker” or combination of markers, as used herein, may refer to any 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 derived from and based on SEQ ID NO: 10 to 21, provided that the modified marker still includes the relevant signature mutation or SNP at the relevant genotype position, which is indicated in SEQ ID NO: 10 to 21 with an ambiguity symbol, thereby uniquely distinguishing the material from other germplasm. Such a modification of a marker may therefore involve a shortening or an elongation of the marker, as long as the nuclear sequence of at least 10 nucleotides before and after the respective signature mutation or SNP is still present. The development of, for example,KASP markers are known as an example of modified markers that can be custom-made based on the marker information provided here, depending on the desired screening or selection method.

[0071] At least two markers, as described herein, can be used to identify a Pisum sativum plant or seed for the production of a plant-based ingredient as defined in any one of claims 1 to 4, wherein the at least one Pisum sativum plant or seed is characterized by at least two markers, each marker identifying a signature SNP mutation, the markers being 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 includes the relevant signature SNP mutation of the one listed under NCIMB No.44401 deposited plant, as shown in Table 6, comprises or encodes, 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 markers, wherein the 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, wherein preferably additionally a BAS-specific marker according to SEQ ID NO: 32, or a modified marker thereof, and / or at least one, at least two or more marker(s) of SEQ ID NO: 10 to 21, or a modified marker thereof, is used.

[0072] In an additional aspect, a method for screening and optionally selecting a plant as the basis for obtaining a plant-based ingredient, preferably a protein ingredient, obtained or obtainable from a Pisum sativum pea, is provided, wherein a representative sample of seeds of the plant has been deposited under NCIMB No. 44401, wherein the plant-based ingredient has an improved taste profile compared to a plant-based ingredient from a pea from a Pisum sativum reference 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 selection includes 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 are used, wherein the 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 includes the relevant SNP signature mutation at the genotype-relevant position, which is indicated in SEQ ID NO: 10 to 21 with a ambiguity symbol, and as shown in Table 6.

[0073] Pisum sativum KWS076 has proven to be homogeneous and stable, as described in the following descriptive information. Pisum sativum KWS076 was self-pollinated over a sufficient number of generations with careful attention to the homogeneity of the plant type and propagated under continued observation of this homogeneity.

[0074] Pisum sativum KWS076 exhibits the following morphological and other characteristics, which are primarily based on field data collected in Wetze, Germany. The characteristics of KWS076 are described below: Ripening level : Number of nodes at the time of first flowering: 13 Height : KWS076 is approximately 70 cm high, which is about 5 cm higher than KWS Exam. vine : Growth form: determined branch 1-2 branches Internodes Zigzag Stocking medium Number of nodes 13 leaflets : Color Green wax Bright Marbled Yes Number of feather pairs re Two stipules miss Flower color : blossom Greenish standard White wing White Kiel White Pods : form Slightly curved Color Green surface Rough length 7 cm End stump Width 8 mm (between the seams) Number of seeds per Sleeve 5 Peas : Color Yellow Seeds (dry, ripe) ): form Around surface Smooth Color pattern Solid color Primary color Yellow Hilum floor paint Light, same color as the base color Color of the cotyledon Yellow grams per 100 seeds n 17

[0075] In one aspect, a Pisum sativum plant seed is provided, preferably for the production of a plant-based ingredient as disclosed herein, or a composition comprising at least one of the plant-based ingredients, wherein the plant or seed is a descendant of a species listed under NCIMB No.44401 deposited seed, optionally by self-pollination 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, each marker identifying a SNP signature mutation, the markers being 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 includes the relevant SNP signature mutation at the relevant position, which is indicated in SEQ ID NO: 10 to 21 with a multi-ambiguity symbol in each case.encoded 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 the 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 includes or encodes the relevant SNP signature mutation as shown in Table 7, preferably using a BAS-specific marker according to SEQ ID NO: 32.

[0076] In another aspect, the use of a Pisum sativum plant or seed for the manufacture of a plant-based ingredient as disclosed herein, or a composition comprising at least one of the plant-based ingredients, is provided, wherein the plant or seed is a descendant of one listed under NCIMB No.44401 deposited seed, optionally by self-pollination or as a first-generation offspring, wherein the at least one Pisum sativum plant or seed is preferably 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, each marker identifying a SNP signature mutation, the markers being 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 includes the relevant SNP signature mutation at the relevant position, which is indicated in SEQ ID NO: 10 to 21 with a multi-ambiguity symbol.encoded 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 the 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 includes or encodes the relevant SNP signature mutation as shown in Table 7, preferably using a BAS-specific marker according to SEQ ID NO: 32.

[0077] In one embodiment, a plant-based ingredient, preferably a protein ingredient, is provided according to the first aspect, wherein the pea plant comprises at least one signature mutation of at least one nucleotide sequence of a gene that affects the saponin biosynthesis pathway in the plant compared to a Pisum sativum reference (synonym: control) plant, wherein the gene encodes for and is selected from a cytochrome P450 monooxygenase (P450), a uridine diphosphate (UDP)-dependent glycosyl transferase (UGT), a TSAR transcription factor, a basic leucine zipper (bZIP) transcription factor, or a squalene synthase, including a cytochrome P450 monooxygenase (P450), a uridine diphosphate-dependent glycosyl transferase (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 part of the plant in which it is expressed, wherein the plant optionally includes at least one further mutation that affects the flavor profile in a gene involved in a pathway other than the saponin pathway, preferably a lipoxygenase (LOX) biosynthesis pathway.

[0078] The present inventors were surprisingly able to identify a specific genetic link between mutation genes, including their non-coding and regulatory regions, that influences the saponin pathway or a LOX pathway in a way that reduces undesirable off-flavors in peas, such that the taste of a plant-based, preferably a protein-based, ingredient of a pea carrying at least one signature mutation of the present invention makes the respective pea line much more advantageous for applications as an ingredient of an alternative food.

[0079] Lipoxygenase (also: LOX EC 1.13.11.12; Linolat: oxygen oxidoreductase) is an iron-containing dioxygenase that catalyzes 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, producing a hydroperoxide with a chain containing a cis-trans conjugated double bond (Grechkin, Recent developments in biochemistry of the plant lipoxygenase pathway; Progress in Lipid Research; 1998). Lipoxygenase activity is known to generate highly reactive compounds such as free radicals, which can react with chlorophylls, carotenoids, ascorbic acid, phenols, and also α-tocopherol (vitamin E) (Gomboeva, Shumaev, Gessler, & Lankin, 2001).LOX plays an important role in the formation of biologically active substances, as it is involved in the biosynthesis of regulators such as jasmonic acid, its methyl ester, abescisic acid and traumatic acid, which play an important role in plant growth and in the 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).

[0080] The products formed during the enzymatic reaction are very important for food quality. Derivatives of hydroperoxides and their degradation products can react with proteins, peptides, and amino acids, which can lead to the formation of off-flavors and a reduction in the nutritional value of the products.

[0081] Consequently, KWS076 is a highly interesting line because it exhibits valuable signature mutations in genes involved in the saponin and / or lipoxygenase pathways, both of which are known to be relevant for the control of off-flavors associated with plant-based ingredients and for the sensory and taste profile of products and compositions derived from them.

[0082] In a further embodiment, a plant-based ingredient, preferably a protein ingredient, is provided, wherein the enhanced taste profile is independently selected from the group consisting of an enhanced taste profile, including a less bitter taste, a less sour taste, a less bean-like taste and / or a less pea-like taste, an enhanced mouthfeel, including a less powdery mouthfeel, a less dull mouthfeel and / or a less coated mouthfeel, a less bitter aftertaste, an enhanced aroma, including a less pea-like aroma and / or a less green / grassy aroma, or any combination thereof.

[0083] Closely related reference or control varieties of Pisum sativum, which are related to the Pisum sativum plant of the present invention, are KWS Kameleon, KWS Exam, and KWS Flam. These varieties are commercially available from KWS Momont SAS, France, and / or KWS SAAT SE & Co. KGaA in Einbeck, Germany. The ZM6 reference genome (see Yang, T., Liu, R., Luo, Y. et al. “Improved pea reference genome and pan-genome highlight genomic features and evolutionary characteristics”, Nat Genet 54, 1553-1563; 2022) can also be used as a reference.

[0084] The seeds of a plant of the Pisum sativum lineage KWS076, synonymously referred to as KM 17AE076, disclosed herein and detailed in the appended claims, were deposited on June 27, 2024, with the National Collections of Industrial, Food and Marine Bacteria (hereinafter “NCIMB”), Wellheads Place, Dyce, Aberdeen, AB21 7GB, Scotland, United Kingdom, and were granted accession number NCIMB 44401 (see form BP / 4). The viability of the material was confirmed by the NCIMB on June 27, 2024 (see form BP / 9). The NCIMB is a recognized international depositary under the Budapest Treaty. This deposit of KWS076 seeds was made under the Budapest Treaty and will be held in the NCIMB vault at least for the duration of the patent's enforceability and will be replaced if the deposit becomes non-viable during that period. Furthermore, the applicant has met all the requirements of 37 CFR sections 1.801-1.809 fulfilled, including the indication of the viability of the sample.

[0085] By performing a deep screen and a bioinformatic comparison, the inventors were able to determine that KWS076 carries several signature mutations, i.e., single nucleotide polymorphism (SNP) mutations, which are present in the KWS076 line but not in a reference line compared to the pea reference genome “ZW6” ​​described in the literature (e.g., in Yang et al.; 2022, supra).

[0086] Mutations in genes, including coding and non-coding regions (e.g., regulatory regions), in KWS076 have been identified and confirmed as the genetic basis for improved sensory performance. Without being bound to any specific theory, it is assumed that these one or more mutations led to a change in the saponin profile of KWS076 peas, and that the resulting protein component from these peas exhibits an improved sensory profile compared to protein from a commercial pea variety.

[0087] In one embodiment according to the various aspects disclosed herein, at least one mutation in the coding region of at least one gene of the saponin and / or LOX pathway in a Pisum plant is provided, generating an early stop codon in the coding sequence that has a beneficial effect on the saponin and / or jasmonic acid and / or abscisic acid and / or traumatic acid profile, wherein an early stop codon may be preferred. Further mutations in other saponin or LOX pathway genes may also play a role in modifying the saponin and / or jasmonic acid and / or abscisic acid and / or traumatic acid profile and the resulting improved sensory performance of a pea protein ingredient derived from KWS076.

[0088] In a further embodiment of the first aspect, the plant-based, preferably pea-based, ingredient is a protein, a starch, a fat, a protein or a combination thereof, preferably a protein.

[0089] In a second aspect, a plant-based composition is provided that includes a plant-based ingredient of any embodiment of the above first aspect.

[0090] In certain embodiments, the plant-based composition may further comprise (i) a second plant-based ingredient derived from a plant that is different from the first plant-based ingredient and / or (ii) a protein ingredient obtained from an animal source, a microbe, a fungus, fermentation and / or a cell culture to obtain a hybrid composition.

[0091] In another embodiment of the second aspect, the composition is a protein composition.

[0092] In a third aspect, an alternative consumer product is provided which includes a plant-based ingredient or plant-based composition according to any embodiment of the first and second aspects.

[0093] In one embodiment, the alternative product is selected from an alternative beverage product, including an alternative milk beverage, an alternative non-milk beverage or an alternative fermented beverage, or from an alternative consumer product, including an alternative dairy product, an alternative seafood product, an alternative egg product and an alternative meat product.

[0094] In another embodiment, the alternative consumer product is a vegetarian or vegan product.

[0095] The seeds of the pea variety KWS076, the plant grown from the seeds, the hybrid pea plant produced by crossing this variety with another pea plant, the hybrid seeds, and various parts of the hybrid pea plant can all be used to obtain plant-based products. Industrial uses include, but are not limited to, use as food for humans, animal feed, and as a raw material in industry.

[0096] As used herein, the term "plant product" refers to the product obtained from or produced by a plant of the pea line KWS076, e.g., the tissues or structures of the KWS076 plant, such as the flower, fruit, seed, leaves, stems, etc., produced by the plant. Furthermore, the pea seeds produced from or derived from KWS076 may be crushed, or a component of the pea seeds may be extracted to obtain a plant extract such as a protein concentrate, protein isolate, texturate, meal, or 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 or jarred, washed, brined, packaged, chilled, frozen, and / or heated pods and / or seeds of the pea plants according to the invention, or any other part thereof. In further embodiments, a processed plant product contains a protein, a sugar or other carbohydrate, a fiber, and / or an aromatic compound extracted, purified, or isolated from the pea plants disclosed herein. In some embodiments, the processed plant product includes washed and packaged pods and / or seeds (or parts thereof) of KWS076, e.g., in a preserved or frozen form.In other embodiments, the processed vegetable product is a whole pod that has been dehydrated and / or baked.

[0097] In certain embodiments, peas from KWS076 can be used to produce pea grits or pea flour. Pea grits or pea flour produced from KWS076 can also be used to produce a pea protein concentrate, a pea protein isolate, and any other form of an ingredient obtained or extracted from the peas.

[0098] In another embodiment, peas from KWS076 can be used to produce various types of "fillers" in food products. Examples of foods containing pea-derived products include protein powders, meat-free burgers / minced meat / sausages, beverages with milk alternatives, yogurt alternatives, vegan cheese and puff pastry, and protein bars. The peas from KWS076 can thus be processed to achieve a similar texture and appearance to many other food products.

[0099] Furthermore, the peas from KWS076 can be used in certain embodiments to produce an intermediate, preferably a protein-based intermediate, which is suitable as an additive or filler in a cosmetic composition or product.

[0100] According to the various embodiments, which here refer to a protein-based ingredient from KWS076 or a homologous line thereof, the high protein content of the peas with a pleasing taste profile is particularly advantageous.

[0101] For consumption, i.e., for food and feed, peas from KWS076 can be advantageously used according to the various aspects and embodiments presented here to produce edible protein ingredients that offer a healthier substitute for animal protein in alternative meats, alternative dairy products, beverages, food supplements, and the like. Peas contain approximately 21.2–32.9% protein, 36.9–49% starch, and 14–26% dietary fiber, based on dry weight (for reference values, see, e.g., Geerts, MEJ, 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 mitigated beany flavour of pea protein isolate,” Food Chemistry. (2019). 278: 665–673; Pietrasik, Z., et al.“Utilization of pea starch and fiber fractions for replacement of wheat crumb in beef burgers,” Meat Science. (October 2019). 161:107974).

[0102] Peas, as legumes, have two aspects that distinguish them from most other food crops. First, they are rich in macro- and micronutrients: they are a good source of protein (rich in essential amino acids such as tryptophan and lysine), slow-digesting carbohydrates, B vitamins, minerals, dietary fiber (soluble and insoluble), phytosterols, and alpha-linolenic acid. They also provide certain amounts of squalene, tocopherols, polyphenols, and triterpenic acids.

[0103] Compared to soybeans or other plant proteins, pea protein is associated with better digestibility and relatively fewer allergenic reactions and negative health controversies.

[0104] However, as described above, there are still challenges in using pea protein as a food ingredient, especially on a large scale, due to organoleptic performance problems, which can be overcome with a pea line from KWS076 or a homologous line.

[0105] In certain embodiments of the provision of a further processed, purified and / or isolated plant-based protein material, the ingredient is in the form of a concentrate, an isolate, a textured product, 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.

[0106] In another embodiment, the plant or plant part from which the ingredient is obtained is selected from a cultivated plant, including soybeans (Glycine max), beans (Phaseolus spp.), broad bean (Phaseolus vulgaris), fava bean (Vicia faba), mung bean (Vigna radiata), pea (Pisum sativum), chickpea (Cicer arietinum), peanut (Arachis hypogaea), lentil (Lens culinaris, Lens esculenta), lupin (Lupinus spp.), white lupin (Lupinus albus), mesquite (Prosopis spp.), carob (Ceratonia siliqua), tamarind (Tamarindus indica), alfalfa (Medicago sativa), cattail (Medicago truncatula), chickweed (Lotus japonicus), licorice (Glycyrrhiza glabra), and clover (Trifolium spp.).), oats, chickpea, kidney bean, maize, potato, wheat, sunflower, rapeseed or rice, wherein the plant or part of the plant from which the ingredient is obtained is preferably a pulse (family Fabaceae, formerly Leguminosae), preferably pea (Pisum sativum), soybean (Glycine max), bean (Phaseolus spp.), including bush bean (Phaseolus vulgaris), and lupins (Lupinus spp.), including white lupin (Lupinus albus).

[0107] A fourth aspect provides a method for producing an alternative consumer product according to the third aspect, comprising: (i) providing a plant-based ingredient or plant-based composition according to the first or second aspect; (ii) adding at least one further additive and / or ingredient; (iii) producing an alternative consumer product.

[0108] In a fifth aspect, the use of a plant-based ingredient or plant-based composition according to one of the first or second aspects for reducing or eliminating off-flavors in an alternative consumer product, preferably in an alternative consumer product as defined in the third aspect, preferably to provide an alternative food, feed or cosmetic composition, optionally in the form of a hybrid product, is provided.

[0109] In a sixth aspect, a method for producing a plant-based ingredient of the first aspect is provided, comprising the following steps: (i) providing a seed of the plant Pisum sativum with a representative sample of seeds deposited under NCIMB No. 44401, or, where appropriate, a progeny thereof; and (ii) extracting a fraction, including a protein fraction, a starch fraction, or a fat fraction, from the pea.

[0110] In one embodiment of the sixth aspect, at least one signature mutation, or more than one signature mutation forming a signature mutation pattern in at least one gene in the saponin and / or LOX biosynthesis pathway, as can be introduced in the Pisum sativum plant with a representative sample of seeds deposited under NCIMB No. 44401, by mutagenesis or genetic engineering, wherein mutagenesis includes chemical mutagenesis, radiation mutagenesis, and genome editing, wherein genome editing includes editing by site-specific nucleases, including zinc finger nuclease (ZFN) systems, transcription activator-like effector nuclease (TALEN) 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.

[0111] In one embodiment, the method produces a protein ingredient in the form of a concentrate, an isolate, a textured product, a powder, a flake, or a flour.

[0112] In another embodiment of the method according to the sixth aspect, the extraction step is a dry extraction method or a wet extraction method.

[0113] In a further embodiment, at least one signature mutation or signature mutation pattern can be introduced into another plant, wherein the plant or plant part is selected from a crop including soybeans (Glycine max), beans (Phaseolus spp.), broad bean (Phaseolus vulgaris), fava bean (Vicia faba), mung bean (Vigna radiata), pea (Pisum sativum), chickpea (Cicer arietinum), peanut (Arachis hypogaea), lentil (Lens culinaris, Lens esculenta), lupin (Lupinus spp.), white lupin (Lupinus albus), mesquite (Prosopis spp.), carob (Ceratonia siliqua), tamarind (Tamarindus indica), alfalfa (Medicago sativa), cattail (Medicago truncatula), chickweed (Lotus japonicus), licorice (Glycyrrhiza glabra), and clover. (Trifolium spp.), oats, chickpea, kidney bean, maize, potato, wheat, sunflower, rapeseed or rice, preferably wherein the plant or part of the plant from which the ingredient is obtained is a pulse (family Fabaceae, formerly Leguminosae), further preferably pea (Pisum sativum), soybean (Glycine max), bean (Phaseolus spp.), including bush bean (Phaseolus vulgaris), and lupins (Lupinus spp.), including white lupin (Lupinus albus).

[0114] In this context, a "site-specific nuclease" refers to a nuclease, or an active fragment thereof, that is capable of specifically recognizing and cleaving DNA at a particular site, the target sequence. Such nucleases typically create 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-inactivated variants thereof.

[0115] A “CRISPR nuclease,” as used herein, is a specific form of site-directed nuclease and refers to any nucleic acid-directed nuclease identified in a naturally occurring CRISPR system, subsequently isolated from its natural context, and preferably modified or combined into a recombinant construct of interest to be suitable as a tool for targeted genome engineering. Any CRISPR nuclease may be used and, if desired, reprogrammed or further mutated to be suitable for the various embodiments according to the present invention, provided that the original wild-type CRISPR nuclease exhibits DNA recognition, i.e., binding properties. CRISPR nucleases also include mutants or catalytically active fragments or fusions of a naturally occurring CRISPR effector sequence or the corresponding sequences encoding it.A CRISPR nuclease can also refer specifically to a CRISPR nickase or even to a nuclease-inactivated variant of a CRISPR polypeptide that has an endonucleolytic function in its natural environment. A large number of different CRISPR nucleases / systems and variants thereof are now known to those skilled in the art and include, among others, CRISPR / Cas systems, including CRISPR / Cas9 systems (EP2771468), CRISPR / Cpf1 systems (EP3009511B1), 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 / CasΦ systems (Pausch et al., Science, 2020, 10.1126 / science. Fig.), CRISPR / CasZ systems and / or any combination, variant, or any 30 catalytically active fragment thereof. A nuclease can be a DNase and / or an RNase, with particular consideration given to the fact that certain CRISPR effector nucleases possess RNA cleavage activity, either alone or in addition to DNA cleavage activity.

[0116] The "guide molecule" or "guide nucleic acid sequence" (usually referred to and abbreviated as guide RNA, crRNA, crRNA+tracrRNA, gRNA, sgRNA, depending on the specific CRISPR system, which is a prototypical nucleic acid-directed site-specific nuclease system) recognizes a target sequence to be cleaved by the nuclease. The 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-directed nuclease binds to form a targeted nuclease complex. The scaffold region may include direct repeats that are recognized and processed by the nucleic acid-directed nuclease to form mature crRNA. A pegRNA may include an additional region within the guide molecule called the "primer-binding site." The "target region" defines the complementarity to the target area that is to be divided.A crRNA, as used here, can therefore be used interchangeably with the term guide RNA if it combines the effects of the now well-established CRISPR nuclease guide RNA functionalities. Certain CRISPR nucleases, e.g., Cas9, can be used by providing two individual guide nucleic acid sequences in the form of a tracrRNA and a crRNA, which may be separate 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 as a single molecule or the sequence encoding it, or as two separate molecules, e.g., crRNA and tracrRNA or the sequences encoding them.

[0117] In certain embodiments, a donor plant or a donor plant population containing one or more alleles associated with an improved trait according to the present invention can be crossed with a recipient plant or a recipient plant population, such as a plant from an elite line or any plant of interest, in order to introduce one or more alleles associated with an improved trait into the recipient plant or plant population, for example, as part of a breeding program. The method according to the invention can be used to identify one or more offspring from such crosses that exhibit an improved trait.

[0118] In certain embodiments, the plants, preferably the peas, produced from these selected plant lines or varieties, preferably pea lines, have a lower or altered saponin content and therefore exhibit less off-flavor perception compared to peas from plants in which the expression of one or more genes of the biosynthesis pathway is not, significantly reduced, or eliminated. The selected pea plants are cultivated, and the peas can be harvested from these plants. The protein ingredient can be extracted from the harvested peas, resulting in a pea protein ingredient that exhibits less off-flavor compared to a control (or reference) pea protein ingredient produced from peas in which the expression of one or more of these genes is not altered (hereinafter referred to as "control peas").

[0119] In certain aspects and embodiments of the method of the sixth aspect, the provision of at least one Pisum sativum plant seed with a gene locus associated with the saponin and / or LOX biosynthesis pathway, which has at least one signature mutation or signature mutation pattern, as can be achieved in the genome of a Pisum sativum plant with a representative sample of seeds deposited under NCIMB No. 44401, compared to a reference or control plant of Pisum sativum that does not carry the at least one signature mutation, or the signature mutation pattern can be achieved temporarily and transiently by reducing the transcription of a gene of the saponin and / or LOX biosynthesis pathway, e.g., by using a knockdown RNAi construct, to achieve the phenotype of a reduced off-flavor of the present invention.

[0120] Details of the invention are described in more detail in the following non-limiting examples. EXAMPLES Example 1: Sensory evaluation of peas: Experimental setup

[0121] Sensory investigations were carried out on peas, pea protein isolates and alternative milk drinks containing such pea protein isolates.

[0122] The sensory dimensions included appearance, aroma, taste, mouthfeel, and aftertaste, and each attribute is summarized in Table 1: TABLE 1 dimension attribute from until definition appearance Color intensity bright dark Measurement of the color intensity of the sample. Particles (on glass) few many Measuring the number of particles on the glass. foam few much Measurement of the product's foam. aroma Total intensity few intensive Measurement of the overall intensity of the product, including all perceptions. pea few intensive Measurement of the intensity of the pea aroma. Muesli few intensive Measurement of the intensity of the grain aroma. Green / Grassy few intensive Measurement of the intensity of the grassy and / or green aroma associated with mowed grass. Sweetish few intensive Measurement of the intensity of the sweet aroma. Sour few intensive Measurement of the intensity of the sour aroma. The taste Total intensity few intensive Measurement of the overall intensity of the product, including all perceptions. pea few intensive Measurement of the intensity of the pea aroma. Muesli few intensive Measurement of the intensity of the grain aroma. Green / Grassy few intensive Measurement of the intensity of the grassy and / or green aroma associated with mowed grass. Milky few intensive Measurement of the intensity of the milky aroma (cow's milk). Süß few intensive Measurement of the intensity of the sweet taste. Sour few intensive Measurement of the intensity of the sour taste. Bitter few intensive Measurement of the intensity of the bitter taste. Salty few intensive Measurement of the intensity of the salty taste. Mouthfeel / Texture Powdery few intensive Measurement of the intensity of the powdery mouthfeel. Dull few intensive Measurement of the intensity of the dull mouth sensation. coating few intensive Measurement of the intensity of the stroking mouth sensation. Astringent few intensive Measurement of the intensity of the astringent mouth sensation. viscosity thin thick Measurement of the intensity of the moist mouth sensation. aftertaste Total intensity few intensive Measurement of the overall intensity of the product, including all perceptions. pea few intensive Measurement of the intensity of the pea aftertaste. Muesli few intensive Measuring the intensity of the grain aftertaste Bitter few intensive Measurement of the intensity of the bitter aftertaste (30 seconds). Süß few intensive Measurement of the intensity of the sweet aftertaste. Sour few intensive Measurement of the intensity of the sour aftertaste. Bitter few intensive Measurement of the intensity of the bitter aftertaste (60 seconds).

[0123] The differences in the various attributes were examined for statistical significance using an analysis of variance (mixed ANOVA) to determine whether the products differed significantly based on an attribute. For the mixed ANOVA, an anchor product is a fixed factor, and the tested products are the random factors, yielding the estimated means and the "least significant difference" (LSD). The LSD represents the minimum difference required, expressed as a scale distance between two products, to conclude that they are significantly different (Fischer's LSD 95%). "Descriptor discrimination" (DD) indicates how often the LSD "fits" into the highest and lowest intensity values ​​of the products. If the DD is greater than 1, the attribute is significantly discriminating between the products. Example 2: Sensory evaluation of peas: Test

[0124] 20 g of cooked and pureed peas from the pea line KWS076, containing a representative sample of seeds deposited under NCIMB No. 44401, were evaluated in comparison to cooked and pureed peas of the commercial pea varieties Flam and Kameleon, available from KWS SAAT SE & Co. KGaA in Einbeck, Germany. A trained sensory panel assessed the peas for taste, mouthfeel / texture, and aftertaste (data not shown, triple measurements not yet completed, initial data very promising). The preliminary data available so far indicate improved mouthfeel and taste, as well as lower overall bitterness and bitter aftertaste, attributed to KWS076 peas compared to the control varieties Kameleon or Flam. Example 3: Sensory evaluation of the protein isolate extracted from peas

[0125] 50 g of protein isolate powder from peas of the pea line KWS076, containing a representative sample of the seeds deposited under NCIMB No. 44401, were tested for their sensory properties in comparison to a protein isolate obtained from the commercially available pea variety Kameleon. A trained sensory panel evaluated 5% protein isolate solutions prepared by combining the protein isolate powders with 1000 mL of water. The results of the evaluation are shown in Table 2 and Fig. 1 shown. TABLE 2: Analysis of mean values ​​(Mean Score): Protein isolate Attribute: Chameleon KWS076 LSD DD Aroma - Overall Intensity 0 -0.445454545 0.445149433 1.000685417 Aroma - Pea 0 -0118181818 0663039253 0.178242567 Aroma - Green / Grassy 0 -0731818182 0770839636 0.949377987 Taste - Sour 0 -0.131818182 0.310832416 0.424081193 Taste - Bitter 0 -0.372727273 0.641130627 0.581359332 Aftertaste - Bitter 30 sec. 0 -0.363636364 0.51689477 0.703501727 Aftertaste - Bitter 60 sec. 0 -0.718181818 0.68798096 1.043897811

[0126] The results of the mean score analysis show a reduction in the aroma, taste, and aftertaste attributes listed in Table 1 for the protein isolate extracted from peas of KWS076 compared to a protein isolate extracted from peas of the commercial variety Kameleon. The results of the mean score analysis show a reduction in the aroma, taste, and aftertaste attributes listed in Table 2 for the protein isolate extracted from peas of KWS076 compared to a protein isolate extracted from peas of the commercial variety Kameleon. The result of this comparison is further detailed in Fig. 1 shown. Example 4: Sensory evaluation of an alternative milk drink

[0127] 200 g of a protein isolate, as described in Example 3, were added to 8,000 mL of a commercial alternative milk base formula provided by VF Nutrition GmbH, trading as vly (registered trademark), Berlin, Germany. A trained sensory panel of 10 participants evaluated the alternative milk drink for its sensory properties, including those listed in Table 3 and in Fig. 2 aroma, taste and aftertaste attributes shown, compared to the same alternative milk base formulation produced using a protein isolate of the commercially available Flam variety. TABLE 3: Analysis of mean scores: Alternative milk drink attribute : Flame KWS076 LSD DD Aroma: Green / Grassy 0.00 -0.58 0.55 1.06 Flavor: Pea 0.00 -0.45 0.45 1 Taste: Bitter 0.00 -0.47 0.36 1.21 Aftertaste: Overall intensity 0.00 -0.33 0.32 1.04 Aftertaste: Bitter for 30 seconds. 0.00 -0.44 0.36 1.21 Aftertaste: Bitter, 60 seconds. 0.00 -0.55 0.37 1.46

[0128] The results of the mean score analysis comparing statistically significant distinguishing features are in Fig.2 graphically represented. These data indicate a significantly improved aroma and taste, as well as a reduced and less intense bitter aftertaste, attributed to the peas of KWS076 compared to the peas of the commercially available Flam variety.

[0129] Although several possible embodiments have been disclosed above, the embodiments of the present invention are not so limited. These exemplary embodiments are not intended to be exhaustive or to unnecessarily restrict the scope of the invention, but were selected and described to illustrate the principles of the present invention so that other people skilled in the art can apply it. Indeed, various modifications of the invention beyond those described herein will be apparent to those skilled in the art from the preceding description. Such modifications are intended to fall within the scope of the appended claims. Example 5: Saponin determination in pea products using LC-MS MS

[0130] To verify that the KWS076 line actually has a favorable saponin profile, comparative liquid chromatography with MS / MS (MRM detection) (LC / MS for short) was performed.

[0131] For LC-MS / MS, 100 mg of each milled sample was mixed with 10 mL of a 70% ethanol:30% dilute-water solution. The mixture was shaken for 30 seconds and then extracted ultrasonically for 30 minutes. The mixture was shaken again for 30 seconds and then centrifuged for 10 minutes at 4,000 rpm and 10°C. 250 µL of supernatant were mixed with 250 µL of acetonitrile and 500 µL of a 90% acetonitrile:10% dilute-water solution. The mixture was shaken again for 30 seconds and filtered through a 0.2 µm wwPTFE syringe filter into a cap. Prior to analysis, 100 µL of the finished extract was mixed with 100 µL of ISTD solution (internal standard) containing soy saponin Ba. For quantification, an external calibration was prepared using the internal standard in a concentration range of 0.1 to 1,500 ng / mL for saponin B in a 50% acetonitrile:14% ethanol:26% dilute aqueous solution. Further dilution was used: 8x.Samples and calibration were performed using an LC-(ESI)-MS / MS method (Agilent HPLC 1290 Inf II with Agilent 6470 Triple Quad detector) on an Agilent Poroshell 120HILIC-Z; 2.1 x 100 mm 1.9 µm column with a gradient of DI water (+0.1% formic acid) and acetonitrile (+0.1% formic acid). MRM transitions used: Soy Saponin Ba (ISTD): 960 → 441 m / z; Saponin B: 944 → 441 m / z; Saponin DDMP: 1070 → 423. The quantification of Saponin DDMP is based on calibration with Saponin B as Saponin B equivalents, as no reference material is available. The results were calculated based on the dry matter (DM) or fresh matter (FM) content.As shown in Table 4 for selected comparative measurements, KWS076 / KM 17AE076 actually exhibits a very favorable saponin profile, since both saponin B and saponin DDMP are significantly reduced compared to reference plants (here: Flam as reference; S in Table 4 used for "saponin"), as shown for the AV (average) saponin values ​​measured below and for the sum of the saponins (B + DDMP; sum of the average). TABLE 4: Comparison of saponin content Average saponin Total saponins ng / mL FM(peeled) ng / mg FM(peeled) ng / mg DM (peeled) ng / mg DM (peeled) ng / mg DM (peeled) genotype SB S DDMP SB S DDMP SB S DDMP SB S DDMP KM17AE076(1) 23,44 21,66 17,72 16,38 19,69 18,20 20,39 18,86 39,25 KM17AE076(2) 24,32 22,51 18,98 17,57 21,09 19,52 15AC082(KWSFlam) (1) 43,36 27,50 32,70 20,74 36,33 23,04 34,92 22,39 57,32 15AC082(KWSFlam) (2) 41,10 26,67 30,17 19,57 33,52 21,75

[0132] Thus, it could once again be confirmed that KWS076 is a highly interesting line for the development of pea lines with reduced saponin levels and therefore reduced off-flavors. Example 6: In-depth evaluation of candidates and determination of SNP markers for signature mutations

[0133] Individual candidates influencing the saponin pathway in peas were then analyzed in more detail in the relevant germplasm of interest and in various comparison lines.

[0134] It is striking that many Pisum sativum candidate genes demonstrably involved in the saponin biosynthesis pathway exhibit only a low degree of identity with homologs / orthologs / paralogs in other cultivated varieties. This is exemplified by the TSAR genes of Pisum sativum in Table 5, which, upon direct matching, show a very low degree of sequence identity / conservation with genes from Chenopodium quinoa. TABLE 5: Exemplary sequence comparison Query and Gene Description Maximum score Total score Query Coverage E-value Percent.Identity Access Psat03G03351 00-T1 TSAR1 Chenopodium quinoaCrop plantRegalonachromosome 6B 65,1 719 45% 8,00E-10 34,75% CP135428.1 Psat04G0244300-T1 TSAR2 Chenopodium quinoaCrop plantRegalonachromosome 6B 60,5 697 51% 5,00E-17 48,28% CP135428.1 Psat04G0244200-T1 Chenopodium quinoaKulturpflanze 59,7 796 50% 7,00E-08 50,00% CP135428.1 TSAR3 RegalonaChromosom 6B

[0135] This revealed that signature mutations for peas of interest must be identified and evaluated on a case-by-case basis, as it is neither meaningful nor successful to extrapolate the results directly from other cultivated varieties. Example 7: Development and application of genetic fingerprint marker sets for identification

[0136] Fingerprint markers were developed for screening for signature mutations and selecting relevant plant material. This development involved analyzing whole-genome sequencing data from the lines in question. The specificity of these markers lies in the careful selection of variants with very low allele frequencies. For a given sample size, these alleles were observed with a frequency of less than or equal to 0.01, meaning they are almost exclusively present in the specified lines. This proves to be an effective method for establishing identity, as it considers the combined probabilities of the observed markers at their respective allele frequencies.

[0137] The application of the marker set for identity determination consists of sequence analyses in the genome regions, regardless of the technology, along each of the different physical positions in the genome, 12 different physical positions for lineage 17AE076, which was deposited under NCIMB No. 44401.

[0138] In order to establish the identity of each line, for each given sequence position with a questionable variant of the reported marker set, the observed allele must match that of the designated SNP assigned to the respective line; only queries with 100% matching genotype calls at each of the given marker positions will be found to have an identity for 17AE076.

[0139] A detailed explanation is provided in Table 6, where light gray cells represent matching genotype calls and thus characteristic SNP signature marker positions between a queried individual and the corresponding genotype calls observed for line 17AE076, registered under NCIMB number 44401. Dark gray cells (and bold and underlined nucleotides of the signature SNP position) indicate discrepancies in genotype calls that lead to loss of identity.

[0140] FP01 to FP12 in Table 6 correspond to SEQ ID NO: 10 to 21.

[0141] If one wishes to enter the production of food ingredients by using germplasm with the advantageous properties and signature mutations of the present invention for downstream (product / food) production, quality checks can be carried out as follows: Typically, one starts with a high-quality reference line that exhibits a phenotype to be improved (or that a customer wishes to have improved). The material is then incorporated into internal routine research as a reference framework for improvements.

[0142] For this purpose, three samples from genetic stocks are typically submitted for inclusion in breeding plans for future generations. Analysis of the stocks in question allows for the identification of lines based on genomic similarities, as revealed here, and further screening using fingerprint marker sets, e.g., as shown in queries 1-3 in Table 6, may follow.

[0143] In the example shown in Table 6, direct comparisons of the observed with the expected genotype calls revealed that one sample, query 1, has a 100% genotype call match with NCIMB line No. 44401, i.e., a 12 / 12 marker match.

[0144] For intelligent and fast screens, fewer markers are sufficient, but usually at least two markers are needed to clearly identify a mismatch.

[0145] This approach enables the direct identification of the genetics underlying NCIMB No. 44401 material and the material derived from it, combined with a functional test for an enhanced taste profile based on the genetics of NCIMB No. 44401.

[0146] Next, further candidates that directly and indirectly influence the saponin biosynthesis pathway were screened. 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 synthase (BAS, SEQ ID NO: 30 and 31) were examined in the corresponding 17AE076 material deposited under NCIMB No. 44401, as mutations were identified that phenotypically influence the plant's flavor profile.

[0147] In particular, certain SNP signature mutations, as shown in Table 7 below, were identified, and markers were designed and tested (SEQ ID NO: 32 to 38) which, alone or in combination, enable the identification of material with a favorable signature mutation. TABLE 7: Further SNP signature mutations in NCIMB No. 44401 Marker Relevante GenotypPosition 17AE076_BAS 1 Marker T 17AE076_Cytochrom Marker 1 A 17AE076_Cytochrom-Marker 2 C 17AE076_Cytochrom-Marker 3 T 17AE076_Cytochrom-Marker 4 T 17AE076_Cytochrom-Marker 5 T 17AE076_Cytochrom-Marker 6 T Quelle 17AE076 NCIMB-Nr.44401

[0148] The results above demonstrate that, based on the analysis of the genome of 17AE076, deposited as NCIMB No. 44401, relevant genotypic and phenotypic insights were gained that are applicable to this specific line as well as to broader contexts: Generally applicable SNP signature mutations and genotype positions were identified that are relevant for the identification and characterization of pea plants with an enhanced flavor profile, which is useful for a variety of food applications. It was shown that signature mutations in at least one gene or a non-coding region thereof, which are directly or indirectly involved in the saponin biosynthesis pathway, are highly characteristic for the rapid and efficient identification of pea plants for the production of plant-based ingredients with an attractive and enhanced flavor compared to plants that do not carry the relevant mutations. QUOTES INCLUDED IN THE DESCRIPTION

[0000] This list of documents cited by the applicant was automatically generated and is included solely for the reader's convenience. The list is not part of the German patent or utility model application. The DPMA accepts no liability for any errors or omissions. Cited patent literature

[0000] EP 2771468

[0115] EP 3009511B1

[0115] WO 2018236548A1

[0115] Cited non-patent literature

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[0079] Yang, T., Liu, R., Luo, Y. et al. „Improved pea reference genome and pan-genome highlight genomic features and evolutionary characteristics“, Nat Genet 54, 1553-1563; 2022

[0083] 27. Juni 2024 bei den National Collections of Industrial, Food and Marine Bacteria (nachstehend „NCIMB“), Wellheads Place, Dyce, Aberdeen, AB21 7GB, Schottland

[0084] Yang et al.; 2022

[0085] 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

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[0101]

Claims

[1] A plant-based ingredient obtained from or derived from a Pisum sativum pea, wherein a representative sample of seeds of this plant has been deposited under NCIMB No. 44401, wherein the plant-based ingredient has an improved taste profile compared to a plant-based ingredient derived from a pea of ​​a Pisum sativum reference 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] Plant-based ingredient according to 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, which affects the saponin biosynthesis pathway in the plant compared to a Pisum sativum reference plant, wherein the gene encodes for and is selected from a cytochrome P450 monooxygenase (P450), a uridine diphosphate (UDP)-dependent glycosyl transferase (UGT), a TSAR transcription factor, a basic leucine zipper (bZIP) transcription factor, or a squalene synthase, including a cytochrome P450 monooxygenase (P450), a uridine diphosphate-dependent glycosyl transferase (UGT), and / or a Triterpene saponin biosynthesis activating regulator (TSAR) transcription factor, including TSAR1, wherein the mutation affects the biosynthesis of saponin in the plant or a part of the plant in which it is expressed,reduced, modified or eliminated, wherein the plant optionally includes at least one further mutation affecting the flavor profile in a gene involved in a pathway other than the saponin pathway, preferably a lipoxygenase biosynthesis pathway, or wherein at least one signature mutation is identified by at least two markers, the markers being 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 includes 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 / or wherein at least one signature mutation is / are 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 the 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 includes or encodes the relevant SNP signature mutation as shown in Table 7. [3] Plant-based ingredient according to claim 1 or 2, wherein the enhanced taste profile is independently selected from the group consisting of an enhanced taste profile, including a less bitter taste, a less sour taste, a less bean-like taste and / or a less pea-like taste, an enhanced mouthfeel, including a less powdery mouthfeel, a less dull mouthfeel and / or a less coated mouthfeel, a less bitter aftertaste, an enhanced aroma, including a less pea-like aroma and / or a less green / grassy aroma, or any combination thereof. [4] Plant-based ingredient according to any of the preceding claims, wherein the ingredient is a protein, a starch, a fat, a protein or a combination thereof, preferably a protein. [5] Plant-based composition comprising a plant-based ingredient according to any one of the preceding claims. [6] Plant-based composition according to claim 5, further comprising: (i) a second plant-based ingredient derived from a plant, which differs from the first plant-based ingredient; and / or (ii) a protein ingredient obtained from an animal source, from a microbe, a fungus, fermentation and / or cell culture to obtain a hybrid composition. [7] Plant-based composition according to claim 5 or 6, wherein the composition is a protein composition. [8] Alternative consumer product comprising a plant-based ingredient or plant-based composition according to any one of claims 1 to 7. [9] Alternative consumer product according to claim 8, wherein the alternative product is selected from an alternative beverage product, including an alternative milk beverage, an alternative non-dairy beverage or an alternative fermented beverage, or wherein the alternative product is selected from an alternative edible product, including an alternative dairy product, an alternative seafood product, an alternative egg product and an alternative meat product. [10] Alternative consumer product according to claim 8 or 9, wherein the product is a vegetarian or a vegan product. [11] Use of a plant-based ingredient or plant-based composition according to any one of claims 1 to 7 for reducing or eliminating off-flavors in an alternative consumer product, preferably in an alternative consumer product as defined in any one of claims 8 to 10. [12] Use of a Pisum sativum plant or seed for the production of a plant-based ingredient according to any one of claims 1 to 4, wherein the plant or seed is registered under NCIMB No. 44401 or is a descendant of the registered plant / seed, preferably wherein the at least one Pisum sativum plant or seed is characterized by at least two markers, each marker identifying a signature SNP mutation, the markers being 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 represents the relevant signature SNP mutation of the plant registered under NCIMB No.44401 deposited plant, as shown in Table 6, comprises or encodes, and / or wherein the at least one Pisum sativum plant seed is characterized by at least one signature mutation identified by at least one, at least two or more markers, wherein the 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, wherein preferably additionally a BAS-specific marker according to SEQ ID NO: 32 or a modified marker thereof, in combination with at least one, at least two or more marker(s) of SEQ ID NO: 10 to 21, or a modified marker thereof, is used. [13] Use of at least two markers for identifying a Pisum sativum plant or seed for the production of a plant-based ingredient as defined in any one of claims 1 to 4, wherein the at least one Pisum sativum plant or seed is characterized by at least two markers, each marker identifying a signature SNP mutation, the markers being 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 includes the relevant signature SNP mutation of the one listed under NCIMB No.44401 deposited plant, as shown in Table 6, comprises or encodes, 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 markers, wherein the 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, wherein preferably additionally a BAS-specific marker according to SEQ ID NO: 32, or a modified marker thereof, and / or at least one, at least two or more marker(s) of SEQ ID NO: 10 to 21, or a modified marker thereof, is used.

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