Pea lines with an improved taste profile of pea-based ingredients

Abstract

The present invention relates to a plant-based ingredient derived from a Pisum sativum line having desired organoleptic properties.

Description

Title of the invention: Pea lines with an improved taste profile of pea-based ingredients. FIELD OF THE INVENTION

[0001] This disclosure relates to plant-based ingredients, in particular protein ingredients, suitable for use in food products, including vegetarian or vegan meat or dairy analogues. The plant-based ingredients are obtained from a new pea strain that gives its ingredients an improved flavor profile. CONTEXT OF THE INVENTION

[0002] The challenges arising from continued population growth, malnutrition and obesity, as well as environmental changes, have generated significant market interest in plant-based ingredients in recent years. Food producers 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, several plant-based protein sources have been considered by food producers, including, but not limited to: soybeans, peas, chickpeas and split peas; lupin; hemp; potato; wheat; cranberries; beans such as white beans, pinto beans, adzuki beans, broad beans, lima beans, black beans, kidney beans and mung beans; pumpkin seeds and seeds of other squashes; and cereals such as rice, sorghum and millet.

[0004] One of the main limitations of working with plant-based ingredients, such as plant-based proteins, as ingredients in food products is the perception of altered flavors. It is possible to mitigate these altered flavors to some extent by using natural flavorings and flavor-masking ingredients. However, the presence of certain aroma- and taste-affecting compounds in plant-based isolates or concentrates makes the product development process laborious, expensive, and time-consuming, and may also be negatively perceived by the consumer.

[0005] Peas, as a source of plant-based ingredients, particularly as a source of protein, have attracted considerable interest from the food industry and consumers in recent years. The presence of an endogenous nitrogen source reduces the soil inputs required for peas and is responsible for their high protein content, making peas an excellent source of protein. plant-based. Moreover, peas, as a source of high-quality protein, have a much lower carbon dioxide footprint (CO2 in g / 100 g of protein produced) than meat-based protein sources or (chicken) eggs (Sustainable Protein Sources. Ist Ed., 2016, Editors: Sudarshan Nadathur, Janitha PD Wanasundara, Laurie Scanlin, eBook ISBN: 9780128027769).

[0006] However, it has been shown that cultivars of Pisum sativum L. differ considerably in their chemical composition, including with regard to their protein, fat, and starch content. Both volatile and non-volatile compounds contribute to the sensory profile of peas and pea-based ingredients, depending on their actual and relative concentrations.

[0007] These differences in chemical composition necessitate specialized processing methods and parameters that may require batch-to-batch customization or continuous process adjustment, thus limiting the potential for large-scale production or use of pea-based ingredients. These differences also result in variations in the attributes of the ingredients and finished products, including organoleptic properties such as taste or flavor, making the use of pea-based ingredients difficult in commercial food production.

[0008] There is therefore a continuing need for pea ingredients with fewer altered flavors and improved taste and organoleptic attributes. Furthermore, there is a great need to define and create suitable pea germplasm and lines that can be cultivated sustainably and that contain high-value, particularly nutritious, plant protein ingredients. Summary of the invention

[0009] According to a first aspect, the invention relates to a plant-based ingredient obtained or obtainable from a Pisum sativum pea, a representative sample of seeds of said plant having been filed under number NCIMB 44382, the plant-based ingredient having an improved taste profile compared with a plant-based ingredient from a pea of ​​a reference Pisum sativum plant, the improved taste profile being due to at least one signature mutation in at least one gene, preferably a signature mutation as indicated in Table 1, including the Psat03G0335100 gene involved in the saponin biosynthesis pathway.

[0010] According to one embodiment of the plant-based ingredient according to the invention, the pea plant comprises at least one signature mutation(s) of at least one nucleotide sequence of a gene affecting saponin biosynthesis in the plant compared with a reference Pisum sativum plant, the encoding gene being selected from a cytochrome P450 monooxygenase (P450), a uridine diphosphate (UDP)-dependent glycosyltransferase (UGT), a TSAR transcription factor, a leucine zipper transcription factor (bZIP), or a squalene synthase, including a cytochrome P450 monooxygenase (P450), a uridine diphosphate-dependent glycosyltransferase (UGT), and / or a triterpene saponin biosynthesis activation regulator (TSAR) transcription factor, including TSAR1, the mutation reducing, altering, or eliminating saponin biosynthesis in the plant or a part of the plant in which it is expressed, optionally, the plant comprising at least one additional mutation affecting the flavor profile in a gene involved in a pathway other than the pathway of the saponin,or at least one signature mutation being identified by at least two markers, said markers being individually selected from a combination of markers from SEQ ID NO: 20 to 30, or a modified marker thereof, provided that the modified marker includes or still codes for the relevant signature SNP mutation of a plant registered under NCIMB number 44382 at the relevant genotypic position indicated in Table 7 and / or at least one signature mutation being identified by at least one, at least two, or more than two marker(s), said marker(s) being individually selected from a combination of markers from SEQ ID NO: 31 to 40, preferably at least SEQ ID NO: 31, or a modified marker thereof, provided that the modified marker includes or still codes for the relevant signature SNP mutation, as indicated in Table 8.

[0011] According to another embodiment of the herbal ingredient according to the invention, the enhanced taste profile is independently selected from the group consisting of an enhanced flavor profile, including a less bitter flavor, a less acidic flavor, a less pronounced bean flavor and / or a less pronounced pea flavor, an enhanced mouthfeel, including a less powdery mouthfeel, a less dull mouthfeel and / or a less coating mouthfeel, a less bitter aftertaste, an enhanced aroma, including a less pronounced pea aroma and / or a less pronounced green / grassy aroma, or any combination thereof.

[0012] According to yet another embodiment, the plant-based ingredient according to the invention is a protein, a starch, a fat or a combination thereof, preferably a protein.

[0013] According to a second aspect, the invention relates to a plant-based composition comprising a plant-based ingredient according to one of the preceding claims.

[0014] According to one embodiment, the herbal composition according to the invention further comprises:

[0015] (i) a second herbal ingredient derived from a plant other than that of the first plant-based ingredient; and / or

[0016] (ii) a protein ingredient obtained from an animal source, a microbe, a fungus, fermentation and / or a cell culture in order to obtain a hybrid composition.

[0017] According to another embodiment, the plant-based composition according to the invention is a protein composition.

[0018] According to a third aspect, the invention relates to a substitute consumable product comprising a plant-based ingredient or a plant-based composition according to the invention.

[0019] According to one embodiment, the substitute consumable product according to the invention is chosen from a substitute beverage product, including a substitute milk beverage, a substitute non-milk beverage or a substitute fermented beverage, or the substitute product being chosen from a substitute consumable product, including a substitute milk product, a substitute seafood product, a substitute egg product and a substitute meat product.

[0020] According to yet another embodiment, the substitute consumable product according to the invention is a vegetarian or vegan product.

[0021] According to a fourth aspect, the present invention relates to a method for producing a substitute consumer product according to the invention, comprising (i) supplying a plant-based ingredient or a plant-based composition according to the invention; (ii) adding at least one additional additive and / or ingredient; (iii) producing a substitute consumer product.

[0022] According to a fifth aspect, the invention relates to the use of an ingredient or plant-based composition according to the invention to reduce or eliminate altered flavors in a substitute consumer product, preferably in a substitute consumer product according to the invention.

[0023] According to a sixth aspect, the invention relates to a process for producing a plant-based ingredient according to the invention, comprising the following steps:

[0024] (ia) the supply of a seed of the plant Pisum sativum having a representative sample of seeds deposited under number NCIMB 44382, or, optionally, of a progeny thereof; or

[0025] (ib) the provision of a seed of the Pisum sativum plant having a Psat03G0335100 gene locus exhibiting a signature mutation pattern as the comparable gene locus in the Pisum sativum plant having a representative sample of seeds registered under NCIMB number 44382, or a Psat03G0335100 gene locus listed in Table 1, preferably, with at least one mutation of the signature mutation pattern being introduced by mutagenesis or genetic engineering, mutagenesis including chemical mutagenesis, radiation-induced mutagenesis and genome editing, genome editing including editing by targeted nucleases, including zinc finger nuclease (ZFN) systems, transcription activator-like effector nuclease (TALEN) systems, meganuclease systems and CRISPR / Cas systems; and

[0026] (ii) the extraction of a fraction, including a protein fraction, a starch fraction or a lipid fraction, from peas, the extraction step preferably being a dry extraction process or a wet extraction process.

[0027] According to one embodiment, the process according to the invention produces a protein ingredient in the form of a concentrate, an isolate, a texturate, a powder, a flake or a flour.

[0028] According to a seventh aspect, the invention relates to the use of a Pisum sativum plant or seed for producing a plant-based ingredient according to the invention, said plant or seed being a seed registered under NCIMB 44382 or being a progeny of a seed registered under NCIMB 44382, optionally by self-fertilization or as first-generation progeny, preferably, at least one Pisum sativum plant or seed being characterized by at least two markers, each marker identifying a signature SNP mutation, said markers being individually selected from a combination of markers from SEQ ID NO: 20 to 30, or a modified marker therefrom, provided that the modified marker includes or still codes for the relevant signature SNP mutation of said plant registered under NCIMB 44382, as shown in Table 7,and / or at least one Pisum sativum plant or seed characterized by at least one signature mutation identified by at least one, at least two, or more than two marker(s), said marker(s) being individually selected from a combination of markers from SEQ ID NO: 31 to 40, or a modified marker thereof, provided that the modified marker includes or still codes for the relevant SNP signature mutation, as indicated in Table 8, preferably a specific TSAR1 marker according to SEQ ID NO: 31, or a modified marker thereof, being used in combination with at least one, at least two, or more than two markers from SEQ ID NO: 20 to 30, or a modified marker thereof.

[0029] According to an eighth aspect, the invention relates to a marker, preferably a set of markers, suitable for screening or selecting a Pisum sativum plant exhibiting an improved taste profile due to at least one signature mutation in at least one gene, preferably a signature mutation as shown in Table 1, comprising the PsatO3GO3351OO gene involved in the saponin biosynthesis pathway, the marker or set of markers comprising at least SEQ ID NO: 31, or a modified marker thereof, alone or in combination with at least one, at least two, or more than two markers as defined previously Brief description of the drawings

[0030] Figure 1 shows a comparative differential diagram of Flam peas and KWS063 peas (synonym: KM 17BP063, filed under the Budapest Treaty as NCIMB 44382), as further detailed in Example IB below. FL stands for flavor, MF stands for mouthfeel, and AT stands for aftertaste.

[0031] Figure 2 shows a differential diagram of a substitute milk drink as further detailed in Example 3 below. AR stands for aroma, FL stands for flavor. BRIEF DESCRIPTION OF THE SEQUENCES

[0032] SEQ ID NO Description 1 TSAR1_ZW6_WGS regulatory 2 TSAR2_ZW6_WGS regulatory 3 TSAR3_ZW6_WGS regulatory 4 TSAR1_CDS 5 TSAR2_CDS 6 TSAR3_CDS 7 TSAR1_protein 8 TSAR2_predicted_protein 9 TSAR3_predicted_protein 10 CDS_Pisum sativum alpha-1,2 mannosyltransferase 11 Protein_Pisum sativum alpha-1,2 mannosyltransferase 12 CDS_Pisum sativum cytochrome P450 72A68-like 1 13 Protein_Pisum sativum cytochrome P450 72A68-like 1 14 CDS_Pisum sativum cytochrome P450 72A68-like 2 15 Protein_ Pisum sativum cytochrome P450 72A68-like 2 16 CDS_ Pisum sativum Terpene cyclase 17 Protein_ Pisum sativum Terpene cyclase 18 CDS_ Pisum sativum beta-amyrin synthase 19 Protein_ Pisum sativum beta-amyrin synthase 20 17BP063_FP_01 21 17BP063_FP_02 22 17BP063_FP_03 23 17BP063_FP_04 24 17BP063_FP_05 25 17BP063_FP_06 26 17BP063_FP_07 27 17BP063_FP_08 28 17BP063_FP_09 29 17BP063_FP_10 30 17BP063_FP_l 1 31 17BPO63_TSAR1 marker 32 17BP063_Cytochrome marker 1 33 17BP063_Cytochrome marker 2 34 17BP063_Cytochrome marker 3 35 17BP063_Cytochrome marker 4 36 17BP063_Cytochrome marker 5 37 17BP063_Cytochrome marker 6 38 17BP063_Cytochrome marker 7 39 17BP063_Cytochrome marker 8 40 17BP063_Terpencyclase marker DEFINITIONS

[0033] In this document, the terms "a," "an," or "the" may refer to one or more. For example, "a" cell may refer to a single cell or a multitude of cells. Furthermore, the term "a plant" may include a plurality of plants.

[0034] A "dry extraction method" includes methods of protein extraction in which the protein fraction is obtained from the starting material by the use of air, pressure, or other non-hydrating processes, or combinations thereof. A "wet extraction method" includes methods of protein extraction in which the protein fraction is obtained from the starting material by the use of water or other solvents, such as acids, bases, combinations of such solvents, etc. The solvents used are preferably food-grade or suitable for use in a food production facility.

[0035] As used in this disclosure, the term "flavor" means a quality detectable by taste and / or smell.

[0036] As used in this disclosure, the term “non-volatile flavor components” refers to molecules that impart flavor to a product but are not volatile in the sense that they cannot be removed by methods such as evaporation. This term applies to both liquids and solids.

[0037] As used in this disclosure, the term "altered flavor" or "altered flavors" refers to an undesirable flavor and refers to the complex interactions of various taste descriptors such as bitter, sour, earthy, bean, salty, sweet, green, savory, nutty, umami, and many others. In addition, other organoleptic parameters such as dryness, dullness, powderiness, moisture and others play a role in the perception of flavor and are encompassed in this document by the expression "altered flavors".

[0038] As used in this disclosure, the term “plant-based ingredient” means any ingredient obtained from plants or parts of plants in a processed form, including protein ingredients, soluble carbohydrate ingredients such as starch, insoluble carbohydrate ingredients such as fiber, lipid ingredients, ash ingredients, etc. A plant-based ingredient may comprise any combination of protein, carbohydrates, and lipids or ash, etc., in any proportion. The term “plant-based ingredient” may be used interchangeably with the term “plant-based food ingredient” in this disclosure.As used in this disclosure, the terms “protein ingredient”, “starch ingredient”, “carbohydrate ingredient”, “lipid ingredient” or “ash ingredient” encompass any form of such ingredient that may be used on its own or in combination with other components of a formula, such as the formula of a herbal composition or a (substitute) food product.

[0039] The plant-based ingredient may be in the form of an isolate, a concentrate, a flour, a texturate, etc.

[0040] The terms “protein texturate” or “textured protein” used in this disclosure refer to an ingredient with structural integrity and an identifiable structure such that individual units, in the form of fibers, shreds, chunks, fragments, granules, slices, and the like, will resist hydration and cooking or other procedures used in the production of food intended for consumption. In general, textured proteins can be used to modify or improve texture and bind water. Edible protein sources from which textured proteins are produced may include, but are not limited to, pulses (e.g., pulse protein), peas, soybeans, corn, wheat, chickpeas, potatoes, rice, sunflowers, etc.Textured proteins may include, but are not limited to, textured pea protein, textured soy flour, textured soy concentrate, textured wheat protein, textured potato protein, or combinations of these proteins. Protein texturization methods are well-known and described in the art, and may include, for example, high-temperature, high-pressure extrusion, spinning, freeze texturizing, chemical or enzymatic texturizing, etc.

[0041] “Protein flour”, as used in this disclosure, refers to an ingredient containing ground peas.

[0042] A “substitute edible product ingredient,” as used in this disclosure, refers to an ingredient derived from a plant, including a protein ingredient, that may be part of an “edible product” or an “edible composition.” Furthermore, at least a portion or fraction of the “substitute edible product ingredient” is suitable as a substitute for a commonly known and commonly manufactured product ingredient, typically as an alternative, for example, to ingredients or foods derived from or produced by animals, including meat, eggs, or dairy products.

[0043] A "food substitute," as used in this disclosure, may be in liquid, semi-liquid, or solid form. In this disclosure, a "food substitute" means a product intended for human or animal consumption that is normally made from ingredients of animal origin, but in which the animal-derived ingredient has been partially or completely replaced by a plant-based substitute ingredient. Non-limiting examples of food substitutes include beverage substitutes, such as a milk substitute or a drinkable yogurt substitute, or food substitutes, such as an egg, beef, dairy, poultry, or seafood substitute.Other alternative food products include pet food or animal feed in which some or all of the animal-derived ingredients are replaced with plant-based ingredients. The replacement or substitution of the animal-derived ingredient may, in some embodiments, be, for example, greater than 70%, greater than 80%, greater than 90%, or greater than 95%. In other embodiments, the replacement or substitution of the animal-derived ingredient may be 70% or less, for example, 60%, 50%, 40%, etc. Alternative food products may also include non-dairy beverages such as sports drinks or smoothies.Food substitutes can also refer to nutritional substitutes, such as herbal powders intended for use as dietary or nutritional supplements.

[0044] A “substitute food” or “alternative nutrition” or a “substitute product / composition (food / cosmetic),” as used in this disclosure, therefore refers to a food, including a liquid food such as beverages, which is generally a plant-based or microorganism-based food that is an alternative to foods of animal origin, including meat or dairy products. Substitute food products may be of particular interest as an alternative source of protein, However, the term "food substitute" refers to any type of essential nutrient, including proteins, carbohydrates, lipids, vitamins, minerals, fiber, and others, that is suitable for food production and is well-accepted, even healthy, as food for humans, livestock, and companion animals. A food substitute product or a substitute feed therefore represents a "consumable substitute product" or an ingredient thereof. The ingredients of the present invention can be used in a food substitute, an animal feed substitute, or a cosmetic substitute product.

[0045] A substitute product or substitute composition, as used in this disclosure, and any pea protein ingredient suitable for their production within the scope of the present invention, is specifically processed (industrially and / or mechanically and / or chemically and / or enzymatically), and a pea protein ingredient of the present invention will generally be processed, isolated, concentrated, and / or otherwise treated for inclusion in a substitute product or composition. Furthermore, a pea protein ingredient of the present invention will generally represent an intermediate ingredient, which has been or can be isolated from a plant representing a part or fraction of a final product or composition, or of a mixture or hybrid product.In a substitute product or composition, the pea protein-based ingredient of the present invention, as a substitute part or fraction of the substitute product or composition, thus replaces a part or fraction that would be present in a commonly known and commonly manufactured product or composition, knowing that it preferably replaces a part or fraction of animal or non-plant origin in the corresponding commonly known and commonly manufactured product.

[0046] In particular, the term "food", as used in this disclosure, means food intended for human consumption, while the term "animal feed", as used in this disclosure, means animal feed.

[0047] In this disclosure, the terms "dairy substitute" or "dairy substitute composition" or "dairy alternative" or "dairy substitute" refer to compositions that mimic the general appearance, nutritional content, and / or taste of dairy products made from animal-derived dairy products without containing animal-derived milk or being substantially free of animal-derived products, and include hybrid products made from laboratory-grown, fermented, and animal-derived components, such as protein components. The dairy substitute may be Completely free of milk or milk proteins of animal origin, or almost free of all milk proteins of animal origin, for example 90% or 95%. The dairy substitute can be a dairy-free cheese, a dairy-free yogurt, a dairy-free ice cream, etc.

[0048] In this disclosure, the terms “meat substitute,” “meat substitute composition,” or “meat alternative” refer to compositions that mimic the general, organoleptic, and / or nutritional properties of consumable products made using any type of meat or meat analogue, including meat, fish, poultry, lab-grown meat products, and fermented meat products. This definition includes hybrid products made from lab-grown, fermented, and animal-derived components, such as protein components. A similar definition is used in this disclosure for “egg substitute,” “egg substitute composition,” and “egg alternative.”Whenever the terms "composition" or "pea protein composition" are used in this disclosure, they refer to a composition of all protein ingredients, flours, concentrates, or isolates described herein, used in an extracted form in combination with other ingredients of different origins to provide said composition. A food or cosmetic substitute is therefore 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 mixed together to provide a basic protein mixture (and, optionally, other additives or ingredients of a different nature and / or origin) comprising pea protein according to the present invention.

[0049] A “consumable product,” as used in this disclosure, refers to goods that are generally considered to be used or consumed in the course of normal business operations, such as food and beverages, office supplies, cleaning and hygiene products, and medical supplies. According to the general concept, there are two main types of consumables: durable consumables, which are expected to last for a long time, and non-durable consumables, which are expected to be consumed relatively quickly. Perishable food and beverages, paper products, ink cartridges, cleaning chemicals, gloves, and syringes are all examples of consumables.The consumable products referred to in this disclosure are non-durable, non-toxic consumable products when swallowed or applied to the human or animal body depending on their intended use, which are generally made of water. less an organic raw material (and possibly others), including foods, beverages, gels, ointments, toothpastes, and the like. The "consumable products / compositions" according to the present invention represent substitute products or compositions comprising at least one plant-based substitute ingredient, preferably a protein ingredient, according to the present invention.

[0050] A “gene,” as used in this disclosure, refers to the coding sequence of a gene, the non-coding sequence, and the upstream and / or downstream regulatory sequences, including an enhancer, an inactivator, promoter elements (e.g., proximal, distal, and central promoter elements), and 5' and / or 3' UTRs. Therefore, a modification of a gene may also include the modification of a non-coding and / or regulatory sequence of that gene.

[0051] A "hybrid composition / product" or a "substitute hybrid composition / product (consumable, including food / cosmetic, etc.)", as used in this disclosure, refers to a product that includes at least partially a "substitute food" or "substitute nutrition" or a "substitute (food) product" comprising a plant-based protein substance according to the present invention, but which may include other ingredients.

[0052] A “knock-down” (inactivation) of a gene refers to an experimental technique by which gene expression (i.e., the transcription of DNA into RNA and thus the amount of active RNA transcripts) is reduced. Reduced expression can, for example, be achieved by silencing gene expression, which reduces or suppresses the transcription rate and thus decreases the amount of available functional RNA.

[0053] A knockout (an inactivation), on the other hand, results in the abolition of expression (transcription / translation), that is, the gene is mistranscribed or not transcribed at all, so that expression is totally abolished or (at the protein level) to such an extent that no functional protein is expressed or is expressed at or near the limit of detection. This can be achieved, for example, by replacing or interrupting the sequence of the target gene. For example, an extra early or premature stop codon, preferably near the start codon, can lead to premature termination of transcription, resulting in the total loss of the functionally translated protein. Alternatively, a knockout can also be achieved by a mutation resulting in a variation of the naturally occurring splice donor or splice acceptor site of a eukaryotic gene comprising exons and introns.The splice donor site typically includes a GU sequence that is almost invariant to . The 5' end of the intron is located within a longer, less 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 (inactivation) as measured by transcription (RNA) or translation (protein) rates. Therefore, the exchange, deletion, or insertion of a single targeted nucleotide can result in a functional knockout, meaning that the sequence encoded by a gene is no longer transcribed or translated. A knockout can also be produced by the deletion of a gene, or a substantial portion of it, at the genomic level, possibly accompanied by a substitution with another sequence.

[0054] A “mutation” or “genome modification” in the context of the present invention means any change to a coherent nucleic acid sequence by modifying a nucleic acid sequence at a given position in the nucleotide sequence, resulting in at least one difference in the (nucleic acid) sequence distinguishing it from the original sequence. In particular, a modification may be effected by the insertion or addition of one or more nucleotides, or by the substitution or deletion of one or more nucleotides in the original sequence, or by any combination of these methods.

[0055] A "nucleic acid construct," a "construct," or an "expression construct" means a nucleic acid molecule encoding or comprising one or more genetic elements, which, once introduced into a target cell, can be transcribed and / or translated into a functional form, for example, RNA, 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 according to the present invention may also be encoded on a set of constructs, which constructs may be introduced into a cell simultaneously or consecutively.

[0056] The terms “RNAi,” “RNA silencing,” or “gene silencing,” as used in this disclosure, refer interchangeably to the process called RNA interference, a mechanism of negative regulation (or knockdown) of genes that has since been demonstrated in all eukaryotes. The mechanism was first recognized and described in plants, where it was called “post-transcriptional gene silencing” or “PTGS.” In RNAi, small RNAs guide specific effector proteins to a target nucleotide sequence by pairing complementary bases, resulting in degradation of the target. A “gene silencing construct” An RNAi agent typically comprises so-called "sense" and "antisense" sequences. Sense and antisense sequences are complementary sequences that are present in opposite directions within a nucleic acid sequence. If a nucleic acid construct includes a sense sequence and a corresponding antisense sequence, the two complementary sequences form a double strand of RNA upon transcription, resulting in an "RNA hairpin." In an RNA hairpin, the sense and corresponding antisense sequences together form a double strand and are separated by an "intron loop intercalating sequence," forming the loop of the hairpin structure.

[0057] The terms "Pisum sativum (L.) plant", "pea plant" and, in short, simply "pea", are used interchangeably in this disclosure, the term "pea" being used in the context of the plant as a whole, but also to refer to parts of it, in particular the seeds / fruits contained in the pea pods.

[0058] A "plant ingredient (from peas)" as used in this disclosure should be understood as the total amount of protein that can be extracted from a fruit or seed (dried or fresh) of the plant (peas). A "protein ingredient (from peas)" should in turn be understood as the total amount of protein in the plant (peas).

[0059] The term "protein concentrate" refers to a protein ingredient with a concentration of between approximately 30% and 60%. A "protein isolate" is an even more concentrated protein ingredient, with a concentration of between approximately 60% and approximately 100%. "Protein meal" refers to the protein that can be obtained directly after dehulling and milling. Since this protein meal is not yet highly processed, it reflects the initial protein content quite directly. Therefore, "protein meal" has also been used by the inventors to define standard ratios when comparing different materials described below. A "protein texturate" or "textured vegetable protein" is used to describe a flour-based product, generally further defatted, that is particularly suitable and used as a meat analogue or meat extender.It is quick to cook and its protein content is comparable to that of some meats. The terms "protein flakes" or "protein powder" also describe the form of the protein. A "protein powder" is generally composed of fine, dry particles produced by grinding, crushing, or disintegrating a solid substance.

[0060] The term "vector" refers to an element used to introduce a nucleic acid construct or a set of nucleic acid constructs into a cellular system. The vector may be a plasmid or a plasmid vector, a cosmid, artificial yeast chromosomes (YAC), bacterial artificial chromosomes (BAC) or PI 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 linear or circular DNA and RNA sequences, or a mixture of such sequences, for introduction or transformation into a plant, plant cell, tissue, organ or material as specified in this disclosure.

[0061] The terms “plant” or “plant cell” or “plant part,” as used in this disclosure, refer to a plant organism, a plant organ, differentiated and undifferentiated plant tissues, plant cells, seeds and their derivatives and progeny. Plant cells include, but are not limited to, for example, cells from seeds, mature and immature cells or organs, including embryos, meristematic tissues, seedlings, callus tissues in various stages of differentiation, leaves, flowers, roots, shoots, male or female gametophytes, sporophytes, pollen, pollen tubes, and microspores and protoplasts, etc.

[0062] “Mutagense” refers to a technique by which modifications or mutations are introduced into a nucleic acid sequence in a random or non-site-specific manner. For example, mutations can be induced by certain chemicals such as EMS (ethyl methanesulfonate) or ENU (N-ethyl-N-nitrosourea) or physically, for example 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, matrix editors, and others. These tools recognize a certain target sequence and allow a modification to be introduced at a specific location within the target sequence.

[0063] A “protein composition” as used in this disclosure refers to a protein isolate that can be obtained directly from a fruit, a seed, in particular a specific pea of ​​the genus Pisum, or to a flour, a protein fraction, or a purified or partially purified protein fraction. The protein composition, depending on its method of preparation, may include denatured and / or partially fragmented proteins, as proteins may have undergone denaturation and / or fragmentation during thermal, chemical, and / or mechanical processing / purification. A protein composition may consist essentially of one protein or a fragment thereof, in particular convicilin, or it may be a mixture of protein compositions including other proteins, in particular globulins and other pea proteins.

[0064] TILLING (Targeting Induced Local Lesions in Genomes) is a process that allows the identification of mutations in a specific gene after (non-specific) mutagenesis has been performed. Mutagenesis can, for example, be carried out using a chemical mutagen such as EMS. Subsequently, a sensitive DNA screening technique is used to identify single-base mutations. The procedures for performing TILLING are known to those skilled in the art.

[0065] A "functional homolog," as used in this disclosure, means a molecule having essentially the same function as a reference molecule. A "structural homolog" means a homolog having a substantial degree of sequence identity with a reference molecule or a portion thereof from which it is derived.

[0066] The term "nucleotide sequence," in the context of the present invention, includes genomic DNA, cDNA, synthetic DNA, and RNA. It preferably means DNA, and more preferably genomic DNA.

[0067] Amino acids are referred to in this disclosure by their amino acid name, their three-letter abbreviation, or their one-letter abbreviation. The term "protein," as used in this disclosure, includes proteins, polypeptides, and peptides. As used in this disclosure, the term "amino acid sequence" is synonymous with "polypeptide" and / or "protein." In some cases, the term "amino acid sequence" is synonymous with "peptide." In some cases, the term "amino acid sequence" is synonymous with the term "enzyme."

[0068] A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in Art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).Thus, a predicted non-essential amino acid residue in a protein according to the invention is preferably replaced by another amino acid residue from the same side-chain family.

[0069] 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 are not related to the functional domains of an enzyme. In another embodiment, the conservative mutations are not related to the catalytic centers of an enzyme.

[0070] In the present disclosure and claims, the conventional one- and three-letter codes for amino acid residues may be used. The three letters code for amino acids, as defined in accordance with the Joint Commission on Biochemical Nomenclature (JCBN) of the IUPACIUB. It is also understood that a polypeptide may code for more than one nucleotide sequence due to the degeneracy of the genetic code.

[0071] A "homologous" plant line or "homologous line" to KWS063, as used in this disclosure, means a line that has been created by modifying or introducing at least one Psat locus as detailed in Table 1 on the basis of this disclosure to modify a gene in the saponin pathway in a targeted manner in accordance with this disclosure, based on the knowledge and teachings provided for KWS063. In addition, genes orthologous to those identified herein may also be derived and used to provide plants with an optimized saponin biosynthesis pathway, for example in other plants of the family Fabaceae, formerly Leguminosae (used interchangeably in this disclosure).

[0072] Where this disclosure relates to the percentage identity of nucleic acid or amino acid sequences with respect to each other, or the identity of a homolog, ortholog, or paralog of a given genetic locus with respect to another, these values ​​define those obtained using the EMBOSS Water Pairwise Sequence Alignments (nucleotide) program (www.ebi.ac.uk / Tools / psa / emboss_water / ) for nucleic acids or the EMBOSS Water Pairwise Sequence Alignments (protein) program (www.ebi.ac.uk / Tools / psa / emboss_water / ) for amino acid sequences. Sequence alignments or comparisons, as used in this disclosure, refer to a full-length alignment of two sequences compared to each other.The tools provided by the European Laboratory of Molecular Biology (EMBL) European Institute of Bioinformatics (EBI) for local sequence alignments use a modified Smith-Waterman algorithm (see www.ebi.ac.uk / Tools / psa / and Smith, TF & Waterman, MS “Identification of common molecular subsequence” Journal of Molecular Biology, 1981 147 (1):195-197). During an alignment, the default parameters defined by EMBL-EBI are used. These parameters are as follows: (i) for amino acid sequences: Matrix = BLOSUM62, gap opening penalty = 10 and penalty. gap extension = 0.5, or (ii) for nucleic acid sequences: Template = DNAfull, gap opening penalty = 10 and gap extension penalty = 0.5. A person skilled in the art knows that, for example, a sequence coding for a protein can be "codon optimized" if the sequence in question is to be used in a different organism compared to the original organism from which the molecule originates.

[0073] The "percentage of protein" or the "percentage of starch" is generally measured by NIR spectrophotometry, but can be measured by other means well known in art.

[0074] As used in this disclosure, the term "plant" includes whole plants, including their offspring or progeny. As used in this disclosure, unless otherwise clearly stated, the term "plant" is intended to refer to a plant at any stage of development. The term "plant part" includes any part or derivative of the plant, including particular plant tissues or structures, plant cells, plant protoplasts, plant cell or tissue cultures from which plants can be regenerated, plant calluses, plant clumps, and intact plant cells in plants or plant parts, such as seeds, kernels, cotyledons, flowers, cotyledons, leaves, stems, buds, roots, root tips, straw, and other similar elements.Plant parts can include processed plant parts or derivatives, including flour, oils, extracts, protein fractions, etc. Examples of "plant parts" include vegetative shoot organs / structures such as leaves, stems, and tubers; roots, flowers, and floral organs / structures, such as bracts, sepals, petals, stamens, carpels, anthers, and ovules; seeds, including the embryo, endosperm, and seed coat; fruits and the mature ovary; plant tissues, such as vascular tissue, background tissue, and others; and cells, such as guard cells, ovule cells, pollen, trichomes, and others; and their offspring. Plant parts may be attached to an intact whole plant or separated from one.These parts of a plant include, but are not limited to, the organs, tissues, and cells of a plant, and preferably the seeds. A "plant cell" is a structural and physiological unit of a plant, comprising a protoplast and a cell wall. The plant cell may be an isolated individual cell or a cultured cell, or part of a more highly organized unit such as, for example, a plant tissue, a plant organ, or an entire plant. "Plant cell culture" means cultures of plant units such as, for example, protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, etc. Ovules, embryo sacs, zygotes, and embryos at various stages of development. “Plant material” means 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 also includes calluses or callus tissues, as well as extracts (such as taproot extracts) or samples. A “plant organ” is a distinct, visibly structured, and differentiated part of a plant, such as a root, stem, leaf, flower bud, or embryo. The term “plant tissue,” as used in this disclosure, means a group of plant cells organized into a structural and functional unit. Any tissue of a plant in planta or in culture is included.This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue cultures, and any group of plant cells organized into structural and / or functional units. The use of this term in connection with, or in the absence of, any specific type of plant tissue as listed above or otherwise included in this definition is not intended to exclude any other type of plant tissue. In some embodiments, the plant part is an organ, tissue, or cell of the plant. In some embodiments, the plant part is a seed, pollen, ovocyte, protoplast, inflorescence, embryo, or callus.

[0075] As used in this disclosure, the terms "control plant" or "control plant part" or "control cell" or "control seed" mean a plant, plant part, plant cell, or seed that has not been subjected to the processes and compositions described in this disclosure. A "control" or "control plant" or "control plant part" or "control cell" or "control seed" serves as a reference point for measuring changes in the phenotype of the plant or plant cell in question. A control plant or plant cell may include, for example: (a) a wild-type plant or cell, that is, one of the same genotype as the starting material of 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 which has been transformed with a null construct (i.e., a construct that has no known effect on the characteristic in question, such as a construct including a marker gene); (c) a plant or plant cell that is an untransformed segregant among the progeny of a plant or plant cell in question; (d) a plant or plant cell genetically identical to the plant or plant cell in question, but which is not exposed to conditions or stimuli (e.g., sucrose) that would induce the expression of a gene in question; (e) the plant or plant cell itself, under conditions in which the; The gene of interest is not expressed. In some cases, a control plant according to this disclosure is grown under the same environmental conditions (e.g., identical or similar temperature, humidity, air quality, soil quality, water quality, and / or pH conditions) as a study plant as described in this disclosure. Similarly, a control protein or control protein composition may refer to a protein or protein composition that is isolated or derived from a control plant. In specific embodiments, a control plant, plant part, or plant cell is a plant, plant part, or plant cell that does not possess a mutated nucleotide sequence in a gene or a regulatory region of a gene as disclosed in this invention.

[0076] The "offspring" includes a pea plant Fl produced from the cross of two pea plants, at least one of which comprises a line, variety or cultivar of pea represented by a seed sample that has been deposited under the terms of the Treaty of Budapest as NCIMB 44382, or crossed with it, and the offspring further includes, but is not limited to, subsequent generational crosses F2, F3, F4, F5, F6, F7, F8, F9 and F10 with the recurring parental line.

[0077] As used in this disclosure with respect to a parameter, the term "decreased," "decreasing," "decreased," "reduced," "lower," or "loss" refers to a detectable negative change (e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%) in the parameter relative to a comparator, such as an established normal or reference level of the parameter, or an established standard control. Accordingly, the terms "decreased," "reduced," and similar terms encompass a partial reduction relative to a control. As used in this disclosure, the terms "elimination," "eliminated," or "discarded" encompass a complete or near-complete reduction relative to a control. For example, this is a negative change of 96%, 98% or 100% of the parameter compared to a control.

[0078] As used in this disclosure, in the context of saponin content or the expression of saponin-related genes, the terms "modification" or "alter" or any variation thereof refer to a modulation of the total saponin content by altering the saponin profile, as described in more detail below. For example, if saponin B levels are reduced, with a concomitant increase in saponin DDMP levels, the total saponin content does not change significantly, but the saponin profile is altered due to changes in the expression of saponin-related genes.

[0079] As used in this disclosure with respect to a parameter, the term "increased" or "growing" or "increase" refers to a positive change detectable (e.g., at least 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) of the parameter relative to a comparator control, such as an established normal or reference level of the parameter, or an established standard control. Accordingly, the terms "increased," "increase," and similar terms encompass a slight, moderate, or significant increase relative to a control. Detailed description of the invention

[0080] The present inventors have detected, characterized and further developed a new line of peas which gives a particularly interesting taste profile to its ingredients and to the substitute food products derived from it.

[0081] In a first aspect, a plant-based ingredient, preferably a protein ingredient, obtained or obtainable from a Pisum sativum pea, a representative sample of seeds of said plant having been filed under number NCIMB 44382, the plant-based ingredient having an improved taste profile compared with a plant-based ingredient from a pea of ​​a reference Pisum sativum plant, the improved taste profile being due to at least one signature mutation in at least one gene, preferably a signature mutation such as shown in Table 1, including the Psat03G0335100 gene involved in the saponin biosynthesis pathway.

[0082] In one embodiment, the signature mutation in at least one gene involved in the saponin biosynthesis pathway is located in a gene, or in a non-coding region thereof, encoding a given enzyme or protein directly involved in the saponin biosynthesis pathway.

[0083] In another embodiment, the signature mutation is part of the germplasm deposited under NCIMB number 44382, the mutation directly or indirectly influencing the saponin biosynthesis pathway.

[0084] In an embodiment of the first aspect, the plant-based ingredient, preferably a protein ingredient, obtained or obtainable from a Pisum sativum pea, in which a representative sample of seeds of said plant has been filed under NCIMB number 44382, carrying at least one signature mutation, can be identified by at least two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve markers, said markers being individually selected from a combination of markers from SEQ ID NO: 20 to 30, or a modified marker therefrom, provided that the modified marker includes or still codes for the relevant SNP signature mutation of a plant filed under the NCIMB number 44382 at the relevant genotypic position indicated in Table 7 and / or in which 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, said markers being individually selected from a combination of markers from SEQ ID NO: 31 to 40, preferably at least SEQ ID NO: 31, or a modified marker of these, provided that the modified marker includes or still codes for the relevant SNP signature mutation, as indicated in Table 8.

[0085] In particular, the combined use of SEQ ID NO: 20 to 30 markers, or modified markers thereof as defined above, and SEQ ID NO: 31 to 40 markers, or modified markers thereof, allows both the precise identification of signature mutations characteristic of NCIMB 44382 and, at the same time, characteristic of the improved taste profile of the relevant herbal ingredient, obtained or obtainable from said pea, or other Pisum sativum material bearing the relevant signature marker scheme as identified in Tables 7 and / or 8 below, alone or in combination, characteristic of the relevant Pisum sativum material according to the present invention.

[0086] Since each of the markers of SEQ ID NO: 20 to 30 comprises at least one signature mutation characterizing or a single nucleotide polymorphism (SNP) as indicated by an IUPAC ambiguity symbol at the relevant genotypic position, as explained in the examples below, said signature mutations being characteristic of NCIMB 44382, these signature mutations allow the precise identification of the relevant plant germplasm, the plant germplasm producing a plant-based ingredient having an improved taste profile compared with a plant-based ingredient from a pea from a reference Pisum sativum plant.

[0087] A “marker” or combination of markers, as used in this disclosure, may refer to any of SEQ ID NO: 20 to 30. 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: 20 to 30, provided that the modified marker still includes the signature mutation or the relevant SNP at the relevant genotypic position indicated by an ambiguity symbol in SEQ ID NO: 31 to 40, respectively, so as to allow unambiguous differentiation of the material from other germplasm. Such a modification of a marker may therefore include a shortening or lengthening of the marker, as long as the core sequence of at least 10 nucleotides upstream and downstream of the respective signature mutation or SNP is still present. Those skilled in the art are well acquainted with the development of, for example, KASP markers, which constitute an example of modified markers that can be custom-designed from the marker information provided in this disclosure, depending on the screening or selection process that is of interest.

[0088] In a further aspect, a method is proposed for screening and, optionally, selecting a plant as a basis for obtaining from said plant a plant-based ingredient, preferably a protein ingredient, obtained or obtainable from a pea of ​​Pisum sativum, a representative sample of seeds of said plant having been filed under number NCIMB 44382, the plant-based ingredient having an improved taste profile compared to a plant-based ingredient from a pea of ​​a reference Pisum sativum plant, the improved taste profile being due to at least one signature mutation in at least one gene, preferably a non-coding region of the gene, the gene being involved in the saponin biosynthesis pathway, the screening and optionally the selection using 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, said markers being individually selected from a combination of markers from SEQ ID NO: 20 to 30, or a modified marker therefrom, provided that the modified marker always includes the signature SNP mutation relevant at the position relevant to the genotype indicated by an ambiguity symbol in SEQ ID NO: 20 to 30, respectively, and as shown in Table 7.

[0089] Pisum sativum KWS063 has demonstrated uniformity and stability, as described in the detailed description below. Pisum sativum KWS063 has been self-pollinated for a sufficient number of generations, carefully maintaining uniformity of plant type, and has been propagated under continuous observation of uniformity.

[0090] Pisum sativum KWS063 exhibits the following morphological and other characteristics, essentially based on field data collected in Wetze, Germany. The characteristics of KWS063 are described below:

[0091] Maturity: Number of nodes at first flowering: 13

[0092] Height: KWS063 measures approximately 70 cm high and 5 cm taller than KWS Exam. Vine:

[0093] Port determined

[0094] Branching 1-2 branches

[0095] Zig-zag internodes

[0096] Medium robustness

[0097] Number of nodes 13 Leaflets:

[0098] Green color

[0099] Light wax

[0100] Marbled Yes

[0101] Number of leaflet pairs two

[0102] Missing stipulations Flower color:

[0103] Greenish venation

[0104] Standard white

[0105] White Wing

[0106] White skittle Pods:

[0107] Slightly curved shape

[0108] Green color

[0109] Rough surface

[0110] Length 7 cm [YES] Frank end

[0112] Width 8 mm (between the sutures)

[0113] Number of seeds per pod 5 Peas:

[0114] Yellow color Seeds (dry, mature):

[0115] Round shape

[0116] Smooth surface

[0117] Monocolor pattern

[0118] Primary color yellow

[0119] Light oil background color, same color as the primary color

[0120] Cotyledon colour yellow

[0121] Grams per 100 of seeds 17

[0122] In one aspect, a Pisum sativum plant or seed is provided, preferably for producing a plant-based ingredient according to any one of claims 1 to 4, said plant or seed being, or being a progeny of, a seed registered under NCIMB 44382, optionally by self-fertilization or as first-generation progeny, preferably, the at least one Pisum sativum plant or seed being characterized by at least two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve markers, each marker identifying a signature SNP mutation, said markers being selected individually from a combination of markers from SEQ ID NO: 20 to 30, or a modified marker therefrom, provided that the modified marker includes or still codes for the relevant signature SNP mutation of said plant filed under NCIMB No. 44382 as shown in Table 7, and / or at least one Pisum sativum plant or seed being characterized by 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 at least eight or more markers, said markers being selected individually from a combination of markers from SEQ ID NO: 31 to 40, or a modified marker therefrom, provided that the modified marker includes or still codes for the relevant signature SNP mutation, as shown in Table 8, preferably, a specific TSAR1 marker according to SEQ ID NO: 31, or a modified marker therefrom, is used in combination with at least one,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 marker(s) of SEQ ID NO: 20 to 30, or a modified marker thereof.

[0123] In another aspect, a use of a Pisum sativum plant or seed is provided for producing a plant-based ingredient according to any one of claims 1 to 4, said plant or seed being or being a progeny of a seed registered under NCIMB 44382, preferably, at least one Pisum sativum plant or seed being 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, at least twelve markers, each marker identifying a signature SNP mutation, said markers being individually selected from a combination of markers from SEQ ID NO: 20 to 30, or a modified marker therefrom, provided that the modified marker includes or still codes for the relevant signature SNP mutation of said plant registered under NCIMB 44382 as shown in Table 7,and / or at least one Pisum sativum plant or seed characterized by 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 at least eight or more markers, said markers being individually selected from a combination of markers from SEQ ID NO: 31 to 40, or a modified marker thereof, provided that the modified marker includes or still codes for the relevant SNP signature mutation, as indicated in Table 8, preferably, a specific TSAR1 marker according to SEQ ID NO: 31, or a modified marker thereof, is used in combination with at least one, at least two, at least three, at least four, at least five, at least six, at the minus seven, minus eight, minus nine, minus ten, minus eleven, minus twelve marker(s) of SEQ ID NO: 20 to 30, or a modified marker of these.

[0124] In yet another aspect, a marker, preferably a set of markers, or the use of a marker or set of markers, is proposed, suitable for screening or selecting a Pisum sativum plant exhibiting an improved taste profile due to at least one signature mutation in at least one gene, preferably a signature mutation such as is shown in Table 1, including the Psat03G0335100 gene involved in the saponin biosynthesis pathway, the marker or set of markers comprising at least SEQ ID NO: 31, or a modified marker thereof, alone or in combination with at least one, at least two, at least three, at least four, at least five, at least six, at least seven or eight markers, or more markers as defined in Tables 7 and 8 and above.

[0125] In one embodiment, a plant-based ingredient, preferably a protein ingredient, of the first aspect is provided, wherein the pea plant comprises at least one signature mutation of at least one nucleotide sequence of a gene affecting saponin biosynthesis in the plant compared to a reference Pisum sativum plant, the encoding gene being selected from cytochrome P450 monooxygenase (P450), uridine diphosphate (UDP)-dependent glycosyltransferase (UGT), TSAR transcription factor, leucine zipper transcription factor (bZIP), or squalene synthase, including cytochrome P450 monooxygenase (P450), uridine diphosphate (UGT)-dependent glycosyltransferase, and / or a saponin biosynthesis activation regulator transcription factor. triterpene (TSAR), including TSAR1, the reducing mutation,modifying or eliminating saponin biosynthesis in the plant or a part of the plant in which it is expressed, possibly the plant comprising at least one additional mutation affecting the flavor profile in a gene involved in a pathway other than the saponin pathway.

[0126] The present inventors have been able, surprisingly, to identify a specific genetic link between mutation genes, including non-coding and regulatory regions thereof, affecting the saponin pathway in a way that reduces undesirable altered flavors in peas, such that the flavor of a plant-based ingredient, preferably a protein ingredient, from a pea carrying at least one signature mutation according to the present invention, makes the respective pea line much more favorable for applications as an ingredient in a food substitute.

[0127] In another embodiment, a plant-based ingredient, preferably a protein ingredient, is provided, in which the improved taste profile is independently selected from the group consisting of an improved taste profile, including a less bitter flavor, a less acidic flavor, a less pronounced bean flavor and / or a less pronounced pea flavor, an improved mouthfeel, including a less powdery mouthfeel, a less dull mouthfeel and / or a less coating mouthfeel, a less bitter aftertaste, an improved aroma, including a less pronounced pea aroma and / or a less pronounced green / grassy aroma, or any combination thereof.

[0128] The reference or control varieties of Pisum sativum closely related to the Pisum sativum plant according to the present invention are KWS Kameleon, KWS Exam, and KWS Flam. Both varieties are commercially available from KWS Momont SAS, France, and / or KWS SAAT SE & Co. KGaA in Einbeck, Germany. Furthermore, the ZM6 reference genome, as described in Table 1, can be used as a reference.

[0129] Seeds of a plant of the KWS063 line of Pisum sativum (synonymously referred to as KM 17BP063), disclosed in this disclosure and referred to in the attached claims, were deposited with the National Collections of Industrial, Food and Marine Bacteria (hereinafter referred to as "NCIMB"), Wellheads Place, Dyce, Aberdeen, AB21 7GB, Scotland, Great Britain, on April 16, 2024, and were assigned NCIMB registration number 44382 (see Form BP / 4). The viability of the material was confirmed by NCIMB on April 17, 2024 (see Form BP / 9). NCIMB is an International Depositary Authority recognized under the Budapest Treaty. This seed deposit KWS063 was made under the Treaty of Budapest and will be kept at the NCIMB filing office for at least the duration of the patent, and will be replaced if the deposit becomes unviable during that period. Furthermore, the applicant has satisfied all the requirements of sections 1.801-1.809 of 37 CFR., including the provision of an indication of the viability of the sample.

[0130] By performing a thorough screening and bioinformatic comparison, the present inventors were able to identify that KWS063 carries several signature mutations, i.e. single nucleotide polymorphism (SNP) mutations present in the KWS063 line, but not in a reference line, namely aligned with the pea reference genome "ZW6" which has been described in the literature, for example in 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). Tableau 1: v ariants de Psat03G0335100: KE_ZW6_hq-vl.chr3 Isoforme Localisation Ori g- Mut. CDS Position Change ment de C DS Change ment de c odon Change ment d'ac ide aminé Psat03G03351 00-T1 271527979 G A 979 327 Caa / Taa Q / * Psat03G03351 00-T1 271528058 C T Psat03G03351 00-T1 271528248 C T Psat03G03351 00-T1 271528251 G A 898 300 Cag / Tag Q / * Psat03G03351 00-T1 271528464 G A 685 229 Caa / Taa Q / * Psat03G03351 00-T1 271528471 C T 678 226 tgG / tgA W / * Psat03G03351 00-T1 271528472 C T 677 226 tGg / tAg W / * Psat03G03351 00-T1 271528542 G A 607 203 Cag / Tag Q / * Psat03G03351 00-T1 271528548 G A 601 201 Cag / Tag Q / * Psat03G03351 00-T1 271528594 C T Psat03G03351 00-T1 271529210 C T Psat03G03351 00-T1 271529300 G A 466 156 Caa / Taa Q / * Psat03G03351 00-T1 271529606 G A 160 54 Caa / Taa Q / * Psat03G03351 00-T1 271529681 G A 85 29 Caa / Taa Q / * Psat03G03351 00-T1 271529705 GA 61 21 Caa / Taa Q / * Psat03G03351 00-T1 271529730 CT Psat03G03351 00-T1 271529812 CT Psat03G03351 00-T1 271529846 CT 3 1 atG / atA M / I

[0131] Mutations, as listed in Table 1, in the KWS063 homolog Psat03G0335100 encoding a transcription factor, have been identified and confirmed in KWS063 as providing a genetic basis for enhanced sensory performance. Without being bound by theory, it is reasonable to assume that this mutation or these mutations resulted in a change in the saponin profile in KWS063 peas, and that the protein ingredient obtained from the peas exhibited an improved sensory profile compared to proteins from a commercial pea variety.

[0132] In one embodiment according to the various aspects disclosed in this disclosure, at least one mutation is provided in the Psat03G0335100 gene, creating an early stop codon in the coding sequence that has a favorable effect on the saponin profile. An example of such a mutation is the G / A substitution described in Table 1 at locus position 271527979, 271528251, 271528464, 271529300, 271529606, 271529681, or 271529705, where an early stop codon may be preferred. Other mutations in other genes of the saponin pathway may also play a role in altering the saponin profile and improving the sensory performance of a pea protein-based ingredient obtained from KWS063.

[0133] In another embodiment of the first aspect above, the plant-based ingredient, preferably pea-based, is a protein, a starch, a fat, a protein or a combination thereof, preferably a protein.

[0134] In a second aspect, a herbal composition is proposed comprising a herbal ingredient of any embodiment of the first aspect above.

[0135] In specific embodiments, the plant-based composition may also include (i) a second plant-based ingredient derived from a plant different from that of the first plant-based ingredient; and / or (ii) a protein ingredient obtained from an animal source, a microbe, a mushroom, fermentation and / or cell culture to obtain a hybrid composition.

[0136] In another embodiment of the second aspect, the composition is a protein composition.

[0137] In a third aspect, a substitute consumable product is proposed comprising a plant-based ingredient or a plant-based composition according to any one of the embodiments of the first and second aspects above.

[0138] In one embodiment, the substitute product is chosen from among substitute beverages, including substitute dairy beverages, substitute non-dairy beverages or substitute fermented beverages, or in which the substitute product is chosen from among substitute consumable products, including substitute dairy products, substitute seafood products, substitute egg products and substitute meat products.

[0139] In another embodiment, the substitute consumable product is a vegetarian or vegan product.

[0140] The seed of the KWS063 pea line, the plant produced from the seed, the hybrid pea plant produced from crossing the variety with any other pea plant, the hybrid seed, and the various parts of the hybrid pea plant can therefore be used to obtain plant-based products. Industrial uses include, but are not limited to, human food, animal feed, and use as raw materials in industry.

[0141] As used in this disclosure, the term "plant product" means the product derived from or produced by a plant of the pea line KWS063, for example, the tissues or structures of the KWS063 plant, such as the flower, fruit, seed, leaves, stems, etc., produced by the plant. Furthermore, pea seeds produced from or derived from KWS063 may be ground, or a component of the pea seeds may be extracted, to include a plant extract such as a protein concentrate, protein isolate, texturate, semolina, flour, and for use in a food or animal feed product.In other embodiments, a processed plant product includes, but is not limited to, the dehydrated, cut, sliced, ground, pureed, dried, baked, fried, canned, jarred, washed, brined, packaged, refrigerated, frozen, and / or heated pods and / or seeds of the pea plants according to the invention, or any other part thereof. In other embodiments, a processed plant product includes a protein, sugar or other carbohydrate, fiber, and / or aromatic compound that is extracted, purified, or isolated from the pea plants described in this disclosure. In these embodiments, the processed plant product includes pods and / or seeds (or parts thereof) of KWS063. washed and packaged, for example in canned or frozen form. In other embodiments, the processed plant product is a whole pod that has been dehydrated and / or cooked.

[0142] In some embodiments, the peas from KWS063 can be used to produce pea semolina or pea flour. The pea semolina or pea flour produced from KWS063 can also be used to produce pea protein concentrate, pea protein isolate, and any other form of ingredient obtained or extracted from peas.

[0143] In another embodiment, the peas from KWS063 can be used to produce various types of "fillers" in food products. Food products containing pea derivatives include, for example, protein powders, meatless hamburgers / minced meat / sausages, dairy-free beverages, yogurt substitutes, vegan cheeses and pastries, and protein bars. Thus, the peas from KWS063 can be processed to produce a texture and appearance similar to those of many other foods.

[0144] Furthermore, in certain embodiments, the peas of KWS063 can be used to produce an intermediate, preferably a protein-based intermediate ingredient, suitable as an additive or filler in a composition or cosmetic product.

[0145] According to the various embodiments relating, in this disclosure, to a protein-based ingredient from KWS063 or a homologous line thereof, the high protein content of peas with a favorable taste profile is particularly advantageous.

[0146] For consumption, i.e. for human and animal food, the peas of KWS063 can be advantageously used according to the various aspects and embodiments presented in this disclosure to produce edible protein ingredients that provide a healthier replacement for animal proteins in meat substitutes, dairy substitutes, beverages, nutritional supplements, etc. Peas contain approximately 21.2 to 32.9% protein, 36.9 to 49% starch, and 14 to 26% dietary fiber, by dry weight (for reference values, see, for example, 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 mitigates 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”, Méat Science, (October 2019). 161:107974). .

[0147] Peas, as legumes, are distinguished from most other food crops in two respects. 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 some amounts of squalene, tocopherols, polyphenols, and triterpenic acids.

[0148] Compared with soybean or other plant-derived proteins, pea protein is associated with greater digestibility and relatively fewer allergic reactions, as well as negative health controversies.

[0149] However, as described above, the use of pea protein as a food ingredient, particularly on a large scale, remains a challenge due to organoleptic performance problems which can be overcome with a pea line from KWS063, or a homologous line.

[0150] In some embodiments intended to provide a processed, purified and / or isolated plant-based protein material, the ingredient is in the form of a concentrate, isolate, texturate, powder, flake or flour, or the ingredient is a protein, a soluble carbohydrate, an insoluble carbohydrate, a lipid, ash, or a combination thereof, preferably a protein.

[0151] In another embodiment, the plant or plant part from which the ingredient is derived, preferably an additional plant-based ingredient, for example for use in a hybrid product or in a substitute product or composition, is obtained or selected from a cultivated plant, in particular soybean (Glycine max), common bean (Phaseolus spp.), field bean (Phaseolus vulgaris), broad bean (Vicia faba), mung bean (Vigna radiata), pea (Pisum sativum), chickpea (Cicer arietinum), groundnut (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), truncated alfalfa (Medicago truncatula), bird's-eye trefoil (Lotus japonicus), licorice (Glycyrrhiza glabra), clover (Trifolium spp.), oats, chickpeas, kidney beans, corn, potatoes, wheat, sunflowers, canola or rice, preferably the plant or plant part from which the ingredient is derived being a legume (family Fabaceae, formerly Leguminosae), more preferably peas (Pisum sativum), soybeans (Glycine max), beans (Phaseolus spp.), including common beans (Phaseolus vulgaris) and lupins (Lupinus spp.), including white lupin (Lupinus albus). .

[0152] In a fourth aspect, a method for producing a substitute consumable product of the third aspect is proposed, comprising (i) supplying a plant-based ingredient or plant-based composition of the first or second aspect; (ii) adding at least one additional additive and / or ingredient; (iii) producing a substitute consumable product.

[0153] In a fifth aspect, it is proposed to use an ingredient or a plant-based composition according to one of the first or second aspects to reduce or eliminate altered flavors in a substitute edible product, preferably in a substitute edible product as defined in the third aspect, preferably to provide a substitute food, animal feed or composition, possibly in the form of a hybrid product.

[0154] In a sixth aspect, a process for producing a plant-based ingredient of the first aspect is proposed, comprising the steps of: (ia) providing a seed of the plant Pisum sativum having a representative sample of seeds filed under number NCIMB 44382, or possibly of a progeny thereof;or (ib) provide a seed of the Pisum sativum plant having a Psat03G0335100 gene locus exhibiting a signature mutation pattern such as the comparable gene locus in the Pisum sativum plant having a representative sample of seeds filed under NCIMB number 44382, or a Psat03G0335100 gene locus listed in Table 1, preferably with at least one mutation of the signature mutation pattern being introduced by mutagenesis or genetic engineering, mutagenesis including chemical mutagenesis, radiation-induced mutagenesis and genome editing, genome editing including editing by targeted nucleases, including zinc finger nuclease (ZFN) systems, transcription activator-like effector nuclease (TALEN) systems, meganuclease systems and CRISPR / Cas systems; and (ii) the extraction of a fraction, including a protein fraction, a starch fraction or a lipid fraction, from the pea.

[0155] In one embodiment, the process makes it possible to produce a protein ingredient in the form of a concentrate, an isolate, a texturate, a powder, a flake or a flour.

[0156] In another embodiment of the process of the sixth aspect, the extraction step is a dry extraction method or a wet extraction method.

[0157] In another embodiment, at least one signature mutation or signature mutation scheme may be introduced into another plant, the plant or part of the plant being selected from a cultivated plant, in particular soybean (Glycine max), bean (Phaseolus spp.), common bean (Phaseolus vulgaris), broad 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), truncated alfalfa (Medicago truncatula), bird's-foot trefoil (Lotus japonicus), licorice (Glycyrrhiza glabra), clover (Trifolium spp.), oat, chickpea, kidney bean, corn, potato, wheat, sunflower, canola, or rice, preferably, the plant or plant part from which the ingredient is derived being a legume (family Fabaceae, formerly Leguminosae), more preferably pea (Pisum sativum), soybean (Glycine max), beans (Phaseolus spp.), including common bean (Phaseolus vulgaris) and lupin (Lupinus spp.), including white lupin (Lupinus albus).

[0158] A “targeted nuclease,” in this disclosure, means a nuclease or an active fragment thereof, capable of specifically recognizing and cleaving DNA at a particular location, the target sequence. These nucleases typically produce a double-strand break (DSB), which is subsequently 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 inactivated nuclease variants.

[0159] A “CRISPR nuclease,” as used in this disclosure, is a site-specific form of nuclease and refers to any nucleic acid-guided nuclease that has been identified in a naturally occurring CRISPR system, subsequently isolated from its natural context, and preferably modified or combined in a recombinant construct of interest for use as a tool for targeted genome engineering. Any CRISPR nuclease can be used and optionally reprogrammed or further mutated to suit the various embodiments according to the present invention, provided that the original wild-type CRISPR nuclease enables DNA recognition, i.e., binding properties. CRISPR nucleases also include mutants, catalytically active fragments, or fusions of natural CRISPR effector sequences, or the respective sequences that encode them.A CRISPR nuclease can also refer, in particular, to a CRISPR nickase or even an inactivated nuclease variant of a CRISPR polypeptide that has an endonucleolytic function in its natural environment. A variety 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 / Cpfl systems (EP3009511B1), and other systems. CRISPR / C2C2, CRISPR / CasX systems, CRISPR / CasY systems, CRISPR / Cmr systems, CRISPR / MAD systems, including, for example, CRISPR / MAD7 (WO2018236548A1) and CRISPR / MAD2 systems, CRISPR / CasO systems (Pausch et al., Science, 2020, 10.1126 / science.abbl400), CRISPR / CasZ systems, and / or any combination, variant, or catalytically active fragment thereof. A nuclease can be an sDNA and / or an sRNA, particularly given that some CRISPR effector nucleases have RNA-degrading activity alone or in addition to DNA-degrading activity.

[0160] The "guide molecule" or "guide nucleic acid sequence" (usually referred to and abbreviated as guide RNA, crRNA, crRNA+tracrRNA, gRNA, sgRNA, depending on the corresponding CRISPR system representing a prototypical nucleic acid-guided site-targeted nuclease system), which recognizes a target sequence to be cut by the nuclease.At least one "guide nucleic acid sequence" or "guide molecule" comprises a "framework region" and a "target region." The "framework region" is a sequence to which the nucleic acid-guided nuclease binds to form a targetable nuclease complex. The frame region may include direct repeats, which are recognized and processed by the nucleic acid-guided nuclease to provide the mature crRNA. A pegRNA may include an additional region within the guide molecule called the "primer-binding site." The "target region" defines the complementarity with the target site, which is intended to be cleaved. A crRNA as used in this disclosure can therefore be used interchangeably with the term guide RNA in the case where it unifies the effects of the now well-established CRISPR nuclease guide RNA functionalities.Some CRISPR nucleases, for example Cas9, can be used by providing two individual guide nucleic acid sequences in the form of a tracrRNA and a crRNA, which can be provided separately or linked by covalent or non-covalent bonds / interactions. The guide RNA can also be a pegRNA from a template rewrite system. The, at least, one guide molecule can be provided as a coherent molecule, or the sequence encoding it, or as two individual molecules, for example crRNA and tracrRNA, or the sequences encoding them.

[0161] In certain embodiments, a donor plant or a population of donor plants, comprising one or more alleles associated with an improved trait according to the present invention, can be crossed with a recipient plant or a population of recipient plants, such as a plant from an elite line or any other plant of interest, in order to introduce one or more alleles associated with an improved trait into the recipient plant or the population of recipient plants, for example, as part of a breeding program. The method according to the present invention can be used to identify one or more offspring of such crosses, exhibiting an improved characteristic.

[0162] In certain embodiments, the plants, preferably peas, produced from these selected plant lines or varieties, preferably peas, have a lower or modified saponin content, and therefore a less pronounced perception of abnormal flavors, compared to peas from plants in which the expression of one or more genes in the biosynthetic pathway is not reduced, 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 with reduced altered flavors compared to a control (or reference) pea protein ingredient made from peas in which the expression of one or more of these genes is not modified (hereinafter referred to as "control peas").

[0163] In certain aspects and embodiments of the method of the sixth aspect, the supply of at least one Pisum sativum plant seed having a Psat03G0335100 gene locus exhibiting a signature mutation pattern as the comparable gene locus in the Pisum sativum plant having a representative sample of seeds filed under NCIMB number 44382, or a Psat03G0335100 gene locus shown in Table 1, can be obtained by using a reference or control plant not carrying at least one signature mutation or signature mutation pattern of the present invention and by using an RNAi knock-down construct based on the SNP mutations shown, among others, in Table 1 to temporarily reduce the transcription of a gene in the saponin pathway in order to obtain a phenotype of an altered flavor according to the present invention.

[0164] The details of the invention are described more precisely in the following non-limiting examples. EXAMPLES

[0165] Example AI: Sensory evaluation of peas: montage

[0166] Sensory evaluations were carried out on peas, pea protein isolates and milk substitute drinks containing such pea protein isolates.

[0167] The sensory dimensions included appearance, aroma, flavor, mouthfeel and aftertaste, and each attribute is summarized in Table 2:

[0168] [Tables2] Dimension Attribute of to Definition Appearance Color intensity bright dark Measures the color intensity of the sample. Particles (on glass) low number Measures the quantity of particles on the glass. Foam low significant Measures the foam of the product. Aroma Overall intensity low intensive Measures the overall intensity of the product, including all perceptions. Pea low intensive Measures the intensity of the pea aroma. Cereal low intensive Measures the intensity of the cereal aroma. Green / grassy low intensive Measures the intensity of the grassy and / or green aroma, associated with freshly cut grass. Sweet low intensive Measures the intensity of the sweet aroma. Acid low intensive Measures the intensity of the acidic aroma. Flavor Overall intensity low intensive Measures the overall intensity of the product, including all perceptions. Pea low intensive Measures the intensity of the pea flavor.Cereal (low intensity): Measures the intensity of cereal flavor. Green / Grassy (low intensity): Measures the intensity of grassy and / or green flavor, associated with freshly cut grass. Dairy (low intensity): Measures the intensity of dairy flavor (cow's milk). Sweet (low intensity): Measures the intensity of sweet taste. Sour (low intensity): Measures the intensity of sour taste. Bitter (low intensity): Measures the intensity of bitter taste. Salty (low intensity): Measures the intensity of salty taste. Mouthfeel / Texture: Powdery (low intensity): Measures the intensity of the powdery mouthfeel. Dull, low, intensive: Measures the intensity of the dull mouthfeel. Coating, low, intensive: Measures the intensity of the coating mouthfeel. Astringent, low, intensive: Measures the intensity of the astringent mouthfeel. Viscosity, thin, thick: Measures the intensity of the moist mouthfeel. Aftertaste: Overall intensity, low, intensive: Measures the overall intensity of the product, including all perceptions. Pea, low, intensive: Measures the intensity of the pea aftertaste. Cereal, low, intensive: Measures the intensity of the cereal aftertaste. Bitter, low, intensive: Measures the intensity of the bitter aftertaste (30 seconds). Sweet, low, intensive: Measures the intensity of the sweet aftertaste. Sour, low, intensive: Measures the intensity of the sour aftertaste. Bitter, low, intensive: Measures the intensity of the bitter aftertaste (60 seconds).

[0169] The differences between the various attributes were analyzed to determine their statistical significance using an analysis of variance (mixed ANOVA), which determines whether the products are significantly differentiated based on an attribute. To perform the mixed ANOVA, an anchor product is a fixed factor and the tested products are the random factors, thus yielding the estimated means and the "smallest significant difference" (hereafter, "LSD"). The LSD corresponds to the minimum difference required, expressed as a scale distance, between two products to conclude that they are significantly different (Fischer's LSD 95%). The "descriptor discrimination" (hereafter, "DD") indicates how often the LSD "matches" the highest and lowest intensity scores appearing among the products. If the DD is greater than 1, the attribute allows for significant discrimination between the products.

[0170] Example AI: Sensory evaluation of peas: tests

[0171] The sensory properties of 20 g of cooked and crushed peas obtained from the KWS063 pea line, with a representative sample of seeds registered under NCIMB number 44382, were evaluated in comparison with cooked and crushed peas from a commercial Flam and Kameleon pea variety, available from KWS SAAT SE & Co. KGaA in Einbeck, Germany. A trained sensory panel assessed the flavor, mouthfeel / texture, and aftertaste of the peas. The evaluation results are presented in Tables 3a and 3b and in [Fig. 1].

[0172] TABLE 3a: Raw score analysis: Crushed peas Attribute: Flam KWS063 LSD DD Flavor - bitter 2.309090909 1.663636364 0.404015202 1.59759965 Flavor - sweet 1.731818182 2.5 0.453049358 1.695580856 Flavor - sour 1.586363636 1.277272727 0.248184676 1.245406904 Mouthfeel - powdery 2.927272727 2.213636364 0.402093456 1.774802231 Mouthfeel - dull 2.504545455 2.122727273 0.336244053 1.135538839 Mouthfeel - coating 2.954545455 2.568181818 0.336273798 1.148955521 Aftertaste - bitter 2.068181818 1.640909091 0.384529342 1.111157669

[0173] TABLE 3b: Analysis of the average score for mashed peas Attribute: Flam KWS063 LSD DD Flavor - bitter 0 -0.645454545 0.404015202 1.59759965 Flavor - sweet 0 0.768181818 0.453049358 1.695580856 Flavor - sour 0 -0.309090909 0.248184676 1.245406904 Mouthfeel - powdery 0 -0.713636364 0.402093456 1.774802231 Mouthfeel - me 0 -0.381818182 0.336244053 1.135538839 Mouthfeel - in robante 0 -0.386363636 0.336273798 1.148955521 Aftertaste - bitter 0 -0.427272727 0.384529342 1.111157669 The results of the mean score analysis comparing the statistically significant differentiating attributes are presented graphically in [Fig. 1]. These

[0174] Data indicate an improvement in mouthfeel and taste, as well as a reduction in overall bitterness and bitter aftertaste, attributed to KWS063 peas compared to Flam control variety peas.

[0175] Example 2: Sensory evaluation of a pea protein isolate

[0176] The sensory properties of 50 g of pea protein isolate powder from the KWS063 pea line, the representative seed sample of which is registered under NCIMB number 44382, were evaluated in comparison to a protein isolate obtained from a commercially available Kameleon pea variety. A trained sensory panel evaluated 5% protein isolate solutions obtained by combining the protein isolate powders with 1000 ml of water. The evaluation results are presented in Table 4.

[0177] TABLE 4: Analysis of the mean protein isolate score Attribute: Kameleon KWS063 LSD DD Aroma - overall intensity 0 -0.09 0.45 0.2 Aroma - pea 0 -0.01 0.66 0.02 Aroma - green / fat 0 -0.15 0.77 0.19 Flavor - sour 0 -0.06 0.31 0.2 Aftertaste - bitter 30 sec 0 -0.04 0.52 0.08 Aftertaste - bitter 60 sec 0 -0.45 0.69 0.65

[0178] The results of the average score analysis indicate reductions in the aroma, flavor, and aftertaste attributes listed in Table 1 for KWS063 pea protein isolate compared to a pea protein isolate extracted from the commercial variety Kameleon. The results of the average score analysis indicate reductions in the aroma, flavor, and aftertaste attributes listed in Table 4 for KWS063 pea protein isolate compared to a pea protein isolate extracted from the commercial variety Kameleon.

[0179] Example 3: Sensory evaluation of a milk substitute drink based on ylyTM

[0180] 200 g of a protein isolate as described in Example 2 were added to 8,000 ml of a commercial milk substitute base formula supplied by VF Nutrition GmbH, trading as vly™, of Berlin, Germany. A trained sensory panel of 10 participants evaluated the sensory properties of the milk substitute, including the aroma, flavor, and aftertaste attributes listed in Table 5 and shown in [Fig. 2], compared to the same milk substitute base formula made with a protein isolate from the commercially available Flam variety.

[0181] TABLE 5: Analysis of the average score: milk substitute drink Attribute: Flam KWS063 LSD DD Aroma: Overall Intensity 0.00 -0.40 0.31 1.28 Aroma: Peas 0.00 -0.55 0.44 1.26 Aroma: Green / Grassy 0.00 -0.67 0.55 1.22 Flavor: Peas 0.00 -0.32 0.45 1.00 Flavor: Sour 0.00 -0.39 0.29 1.34 Flavor: Bitter 0.00 -0.46 0.39 1.21 Aftertaste: Overall Intensity 0.00 -0.22 0.32 1.04 Aftertaste: Bitter 30 Sec 0.00 -0.21 0.36 1.21 Aftertaste: Bitter 60 Sec 0.00 -0.22 0.37 1.46

[0182] The results of the mean score analysis comparing the statistically significant differentiating attributes are presented graphically in [Fig. 2]. These data indicate a significant improvement in aroma and flavor, as well as a reduced and less intense bitter aftertaste, attributed to KWS063 peas compared to commercial Flam peas.

[0183] Although several possible embodiments are disclosed above, the embodiments of the present invention are not so limited. These examples of embodiments are not intended to be exhaustive or to unnecessarily limit the scope of the invention, but have been selected and described to explain the principles of the present invention in such a way that other persons skilled in the art may carry it into practice. Indeed, various modifications of the invention, in addition to those described in this disclosure, will become apparent to specialists in the light of the preceding description. Such modifications fall within the scope of the appended claims.

[0184] Example 4: Further evaluation of candidates

[0185] Individual candidates influencing the saponin pathway in pea were then further analyzed in the relevant germplasm of interest and in various comparative lines.

[0186] It is striking that many candidate genes of Pisum sativum involved in the saponin biosynthesis pathway show only a low level of identity with homologs / orthologs / paralogs in other cultivars. This is shown as an example for the TSAR genes of Pisum sativum in Table 6, which demonstrates that it will be almost impossible to extrapolate results from different

[0187] cultivated to candidate genes of Pisum sativum, because the genes, including TSAR1, TSAR2 and TSAR3, generally exhibit a very low degree of sequence identity. TABLE 6: Example of sequence comparison Query and gene Description Maximum score Early score Query coverage E value Per. i dent. Record Psat03G03 35100-T1 TSAR1 Cultivar Chenopodium quinoa Regalona chromosome 6B 65.1 719 45% 8.00E- 10 34.75% CP135428. 1 Psat04G02 44300-T1 TSAR2 Cultivar Chenopodium quinoa Regalona chromosome 6B 60.5 697 51% 5.00E- 17 48.28% CP135428. 1 Psat04G02 44200-T1 TSAR3 Cultivar Ch enopodium qu inoa Regalona chromosome 6B 59.7 796 50% 7.00E- 08 50.00 % CP135428. 1 From the above, it was deduced that signature mutations of interest should be identified and evaluated on a case-by-case basis, since it was neither significant nor fruitful to directly extrapolate the analysis results obtained from other cultivars.

[0188]

[0189]

[0190]

[0191] Example 5: Development and application of genetic fingerprinting marker series for identity establishment Fingerprint markers have been developed to detect signature mutations and select relevant plant material. This development involved analyzing whole-genome sequencing data from lines of interest. The specificity of these markers lies in the careful selection of variants with very low allele frequencies. Within a given sample size, these alleles were observed at frequencies less than or equal to 0.01; in other words, they are almost exclusively present in the lines in question. This proves to be an effective way to establish identity, as it takes into account the combined probabilities of the observed markers at their respective allele frequencies. Applying the set of markers to establish identity involves sequence analyses in genomic regions, regardless of the form of the technology, along each of the different physical positions of the genome, 12 different physical positions for the 17BP063 line filed under NCIMB number 44382.

[0192] For the identity of each lineage to be established, for each given sequence position with a variant in question of the reported marker set, the observed allele must correspond to that of the denoted SNP assigned to the respective lineage, only queries with 100% of genotype calls corresponding to each of the given marker positions are established with an identity of 17BP063.

[0193] A detailed explanation is illustrated below in Table 7, where light grey cells correspond to matching genotype calls and thus characterize the positions of signature SNP markers between an interviewed individual and the corresponding genotype calls observed for line 17BP063 filed under NCIMB number 44382. Dark grey cells (and the signature SNP position nucleotides marked in bold and underlined) indicate mismatches in genotype calls, resulting in a loss of identity.

[0194] FP01 to FP11 in Table 7 correspond to SEQ ID NO: 20 to 30, respectively.

[0195] If it is now desired to begin producing food ingredients using germplasm exhibiting the favorable characteristics and signature mutations according to the present invention for downstream production (product / food), quality controls can be carried out as follows: generally, one starts with a high-quality reference line having a phenotype to be improved (or that a customer wishes to see improved). The material will then be included in routine internal research as a reference framework for improvement.

[0196] To this end, three samples of genetic strains are generally submitted for inclusion in breeding schemes for future generations. After analyzing the strains in question, genomics makes it possible to identify the similarities between the lines as disclosed here and to proceed with a more in-depth screening using sets of fingerprinting markers, for example as indicated in queries 1 to 3 of Table 7.

[0197] In the example shown in Table 7, direct comparisons between observed and expected genotype calls showed that one sample, Query 2, has a 100% genotype call match with NCIMB line No. 44382, i.e. a marker match of 11 / 11.

[0198] For smart and fast screens, it is sufficient to have fewer markers, but generally at least two markers, to unambiguously identify a non-match.

[0199] This approach allows for direct identification of the genetics underlying NCIMB material No. 44382 and material derived from it, in combination with a functional test for an improved taste profile resulting from NCIMB genetics no. 44382.

[0200] A party interested in developing a pea-derived material with improved taste characteristics, using the registered lines and teachings provided in this disclosure, may wish to implement a production protocol for a dairy substitute ingredient. To ensure due diligence, it is necessary to verify that the lines in question undergo routine quality control prior to large-scale production and implementation of the protocol. To this end, the genetic identity of the material should first be established at the production facilities using random samples from several production batches, for example, queries 1 to 3 in Table 7.

[0201] After testing more than 30 independent fingerprint markers, via a preferred genotyping platform, we can perform a direct comparison of expected genotype calls against observed genotype calls at each position for each of the samples, all based on the reference to material 17BP063 already confirmed to have significant SNPs in the saponin biosynthesis pathway that positively influence taste.After careful evaluation of all 30 independent fingerprinting markers, we determined that, while there are similarities between several of the expected genotype appeals from multiple production batches and the reference framework, including a highly specific TSAR1 marker for one of the taste-enhancing signature mutations in the 17BP063 line, none of the three random samples share exactly the same genotype appeals as the expected alleles across all markers. Based on these observations, we can exclude germplasm from queries 1 and 3, ensuring that all genetic strains undergoing mass production and protocol implementation have the same genetic basis as a starting point for their expected phenotypic performance.

[0202] The most effective relevant markers FP_01 to FP_11 (SEQ ID NO: 20 to 30) are highly specific and unambiguously reveal material 17BP063 alone or in appropriate combinations and can be used for screening pea lines with the other markers detailed below.

[0203] TABLE 7: SNP signature mutations within NCIMB 44382 MARKER Relevant position of genotype Query 1 Query2 Query3 17BP063_FP_01 YYYY 17BP063_FP_02 T Ç TT 17BP063_FP_03 YYYY 17BP063_FP_04 CGCC 17BP063_FP_05 GGGG 17BP063_FP_06 TTTT 17BP063_FP_07 AGAA 17BP063_FP_08 CCCC 17BP063_FP_09 AAAI 17BP063_FP_10 CTCC 17BP063_FP_ll TTTT 17BPO63_TSAR1 GGGA Identity 17BP063 N CIMB n° 4 4382 Not established 17BP063 Not established

[0204]

[0205]

[0206] Next, other candidates that directly and indirectly influence the saponin biosynthesis pathway were selected. Pisum sativum TSAR 1 to 3 (SEQ ID NO: 1 to 9), Pisum sativum alpha-1,2 mannosyltransferase (SEQ ID NO: 10 and 11), two variants of Pisum sativum cytochrome P450 72A68-like (SEQ ID NO: 12 to 15), Pisum sativum terpene cyclase (SEQ ID NO: 16 and 17), and Pisum sativum-specific beta-amyrin synthase (BAS, SEQ ID NO: 18 and 19) were evaluated in the relevant material 17BP063 filed under NCIMB number 44382, as mutations were identified as having a phenotypic influence on the taste profile of the plant. In particular, certain signature SNP mutations, as shown in Table 8 below, have been identified and markers have been designed and tested (SEQ ID NO: 31 to 40) which, alone or in combination, allow the identification of material with a favorable signature mutation. TABLE 8: Other SNP signature mutations within NCIMB #44382 Marker Relevant position of genotype 17BPO63_TSAR1 marker G 17BP063_Mannosyltransferase 1 A 17BP063_Mannosyltransferase 2 T 17BP063_Mannosyltransferase 3 T 17BP063_Mannosyltransferase 4 T 17BP063_Mannosyltransferase 5 T 17BP063_Mannosyltransferase 6 A 17BP063_Mannosyltransferase 7 T 17BP063_Mannosyltransferase 8 A 17BP063_Terpencyclase marker G Source 17BP063 NCIMB #44382

[0207] The above results show that, based on the analysis of the genome of 17BP063 filed as NCIMB No. 44382, relevant genotypic and phenotypic conclusions could be made, which are applicable to the specific line, but also beyond: signature SNP mutations and generally applicable genotypic positions could be identified, which are relevant for the identification and characterization of pea plants with an improved taste profile useful for a variety of food applications,Given that it has been demonstrated that signature mutations in at least one gene or a non-coding region thereof, directly or indirectly involved in the saponin biosynthesis pathway, are highly characteristic for rapidly and efficiently identifying 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.

[0208] It is interesting to note that a mutation in the TSAR1 gene has been identified and that it significantly influences the saponin biosynthesis pathway and, in turn, leads to a decrease in the levels of altered flavors in the plant material concerned. This marker is therefore a reasonable tool for screening for comparable mutations in the pea germplasm.

Claims

Demands

1. Herbal ingredient, characterized in that it is obtained or is capable of being obtained from a Pisum sativum pea, a representative sample of seeds of said plant having been filed under NCIMB 44382, said ingredient having an improved taste profile compared with an herbal ingredient derived from a pea of ​​a reference Pisum sativum plant, the improved taste profile being due to at least one signature mutation in at least one gene, preferably a signature mutation as indicated in Table 1, including the Psat03G0335100 gene involved in the saponin biosynthesis pathway.

2. Herbal ingredient according to claim 1, characterized in that the pea plant comprising at least one signature mutation(s) of at least one nucleotide sequence of a gene affecting saponin biosynthesis in the plant compared with a reference Pisum sativum plant, the encoding gene being selected from cytochrome P450 monooxygenase (P450), uridine diphosphate (UDP)-dependent glycosyltransferase (UGT), TSAR transcription factor, leucine zipper (bZIP) transcription factor, or squalene synthase, including cytochrome P450 monooxygenase (P450), uridine diphosphate-dependent glycosyltransferase (UGT), and / or triterpene saponin biosynthesis activation regulator (TSAR) transcription factor, including TSAR1, the reducing mutation,modifying or eliminating saponin biosynthesis in the plant or a part of the plant in which it is expressed, optionally, the plant comprising at least one additional mutation affecting the taste profile in a gene involved in a pathway other than the saponin pathway, or at least one signature mutation being identified by at least two markers, said markers being individually selected from a combination of markers from SEQ ID NO: 20 to 30, or a modified marker of these, provided that the modified marker includes or still codes for the relevant signature SNP mutation of a plant filed under NCIMB No. 44382 at the relevant genotypic position indicated in the table, 7 and / or at least one signature mutation being identified by at least one, at least two, or more than two marker(s), said marker(s) being individually selected from a combination of markers from SEQ ID NO: 31 to 40, preferably at least SEQ ID NO: 31, or a modified marker of it / these, provided that the modified marker still includes or codes for the relevant SNP signature mutation, as indicated in Table 8.

3. Herbal ingredient according to claim 1 or 2, characterized in that the enhanced taste profile is independently selected from the group consisting of an enhanced flavor profile, including a less bitter flavor, a less acidic flavor, a less pronounced bean flavor and / or a less pronounced pea flavor, an enhanced mouthfeel, including a less powdery mouthfeel, a less dull mouthfeel and / or a less coating mouthfeel, a less bitter aftertaste, an enhanced aroma, including a less pronounced pea aroma and / or a less pronounced green / grassy aroma, or any combination thereof.

4. Plant-based ingredient according to any one of the preceding claims, characterized in that the ingredient is a protein, a starch, a fat or a combination thereof, preferably a protein.

5. Herbal composition, characterized in that it comprises an herbal ingredient according to any one of the preceding claims.

6. Herbal composition according to claim 5, characterized in that it further comprises: (i) a second herbal ingredient derived from a plant different from that of the first herbal ingredient; and / or (ii) a protein ingredient obtained from an animal source, a microbe, a fungus, fermentation and / or cell culture to obtain a hybrid composition.

7. Herbal composition according to claim 5 or 6, characterized in that the composition is a protein composition.

8. Substitute consumable product comprising a plant-based ingredient according to any one of claims 1 to 4 or a plant-based composition according to any one of claims 5 to 7.

9. Substitute consumable product according to claim 8, characterized in that the substitute product is chosen from a substitute beverage product, including a substitute milk beverage, a substitute non-milk beverage or a substitute fermented beverage, or the substitute product being chosen from a substitute consumable product, including a substitute milk product, a substitute seafood product, a substitute egg product and a substitute meat product.

10. Substitute consumable product according to claim 8 or 9, characterized in that the product is a vegetarian or vegan product.

11. A method for producing a substitute consumable product according to any one of claims 8 to 10, characterized in that it comprises (i) supplying a plant-based ingredient according to any one of claims 1 to 4 or a plant-based composition according to any one of claims 5 to 7; (ii) adding at least one additional additive and / or ingredient; (iii) producing a substitute consumable product.

12. Use of an ingredient according to any one of claims 1 to 4 or of a plant-based composition according to any one of claims 5 to 7 to reduce or eliminate altered flavors in a substitute consumer product, preferably in a substitute consumer product as defined in any one of claims 8 to 10.

13. A process for producing a plant-based ingredient according to claims 1 to 4, characterized in that it comprises the following steps: (ia) supplying a seed of the plant Pisum sativum having a representative sample of seeds filed under NCIMB 44382, or, optionally, a progeny thereof; or (ib) supplying a seed of the plant Pisum sativum having a gene locus Psat03G0335100 exhibiting a signature mutation pattern such as the comparable gene locus in the plant Pisum sativum having a representative sample of seeds filed under NCIMB 44382, or a gene locus Psat03G0335100 listed in Table 1, preferably, at least one mutation of the signature mutation pattern being introduced by mutagenesis or by genetic engineering, the mutagenesis comprising

14.

15. chemical mutagenesis, radiation-induced mutagenesis, and genome editing, genome editing including editing by targeted nucleases, including zinc finger nuclease (ZFN) systems, transcription activator-like effector nuclease (TALEN) systems, meganuclease systems, and CRISPR / Cas systems; and (ii) the extraction of a fraction, including a protein fraction, a starch fraction or a lipid fraction, from peas, the extraction step preferably being a dry extraction process or a wet extraction process. A process according to claim 13, characterized in that the process produces a protein ingredient in the form of a concentrate, isolate, texturate, powder, flake, or flour. Use of a Pisum sativum plant or seed to produce a plant-based ingredient according to any one of claims 1 to 4, characterized in that said plant or seed is a seed registered under NCIMB 44382 or a progeny of a seed registered under NCIMB 44382, optionally by self-fertilization or as first-generation progeny, preferably, at least one Pisum sativum plant or seed being characterized by at least two markers, each marker identifying a signature SNP mutation, said markers being individually selected from a combination of markers from SEQ ID NO: 20 to 30, or a modified marker thereof.provided that the modified marker includes or still codes for the relevant signature SNP mutation of said plant filed under NCIMB number 44382, as indicated in Table 7, and / or at least one Pisum sativum plant or seed being characterized by at least one signature mutation identified by at least one, at least two, or more than two marker(s), said marker(s) being individually selected from a combination of markers from SEQ ID NO: 31 to 40, or a modified marker thereof, provided that the modified marker includes or still codes for the relevant signature SNP mutation, as indicated in Table 8, preferably a specific TSAR1 marker according to SEQ ID NO: 31, or a modified marker thereof, being used in combination with at least one, at least, two, or more than two markers of SEQ ID NO: 20 to 30, or a modified marker of this / these.

16. Marker, preferably set of markers, suitable for screening or selecting a Pisum sativum plant exhibiting an improved taste profile due to at least one signature mutation in at least one gene, preferably a signature mutation as shown in Table 1, characterized in that it comprises the Psat03G0335100 gene involved in the saponin biosynthesis pathway, the marker or set of markers comprising at least SEQ ID NO: 31, or a modified marker thereof, alone or in combination with at least one, at least two or more than two markers as defined in claims 2 or 15.

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