Pea protein isolation with organic acids

Abstract

The present invention is directed to methods for providing a plant-based protein ingredient having improved physicochemical properties, including vegetarian or vegan meat or dairy analogues. The protein ingredients have improved physicochemical properties stemming from improved methodologies in the downstream processing of protein isolates.5

Description

[0001] Munich, 27 March 2026

[0002] Our Ref.: KM 5682-02WO CMC / MCL

[0003] Applicant: KWS SAAT SE & Co. KGaA

[0004] Serial Number: New Application

[0005] KWS SAAT SE & Co. KGaA

[0006] GrimsehlstraBe 31, 37574 Einbeck, Germany

[0007] Pea protein isolation with organic acids

[0008] Technical Field

[0009] The present invention is directed to methods for providing a plant-based protein ingredient having improved physicochemical properties, including vegetarian or vegan meat or dairy analogues. The protein ingredients have improved physicochemical properties stemming from improved methodologies in the downstream processing of protein isolates.

[0010] Background

[0011] In view of an increasing consumer awareness regarding animal welfare and diet-associated illnesses, including obesity, type II diabetes, prevention of dementia and cardiovascular diseases, the development of healthier food products is of increasing interest and there is an ever-increasing consumer demand for plant-based food and food additives. In view of challenges related to climate change and a steady increase in the world population further increase the awareness related to ecological sustainability in the context of developing new seeds, planting and using the same.

[0012] Producers are thus facing the challenges of providing plant-based alternatives at a scale that meets consumers’ expectations in taste, texture, and other functional and organoleptic properties. Especially, the nutritional profile and physicochemical properties are important to the consumers when selecting an alternative food.

[0013] Furthermore, the attention of customers toward health impacting food rises. Thus, for being accepted as a consumable, the product needs to have e.g. a valuable fatty acid profile, is low in sodium chloride or contain important nutrients.

[0014] To meet the aforementioned demands of consumers, several sources of plant-based proteins have been considered by producers, including but not limited to: soybeans; peas,chickpeas and split peas; cranberries; beans such as navy, pinto, adzuki, fava, lima, black, red kidney, and mung beans; pumpkin seed and seed from other squash; and grains such as rice, sorghum, and millet. Consumers look for clean-labelled, locally-sourced and sustainable ingredients, which factors are also considered by producers. Further, certain consumers do not wish to consume animal products, including milk from cows, at all in view of its animal origin, due to lactose intolerance, or due to dairy allergies and the like. They may also see potential environmental sustainability issues. The global consumable industry has a continuing need to find plant-based protein sources that meet these requirements and preferences.

[0015] Peas (Pisum sativum or Pisum sativum L.) are annual plants with a one-year life cycle belonging to the family of Leguminosae. Peas belong to the oldest cultivated crops and domestication of wild pea plants likely already began with the start of agriculture about 11 ,000 years ago. Pea plants contain symbiotic bacteria within their root system with the special ability to fix nitrogen from the atmosphere, making them a highly attractive source of protein which meets the preferences of locally grown, sustainable agricultural practices. One major challenge of working with pea plants, especially when it comes to the production of food alternatives is the fact that to date, pea protein isolate as natural product suffers from severe batch-to-batch variations. Thus, methods are needed to achieve lesser batch-to-batch variations to yield pea protein isolates with the same and compared to conventionally used methods improved physicochemical properties. Furthermore, current methods known from the state of the art have a high impact on the sodium chloride content and salty taste of a pea protein ingredient. Thus, for incorporating this into a final application, such as an alternative food or meat product, the taste needs to be adjusted by e.g. adding additional flavoring and active substances. In terms of a “clean label”, which should be achieved for meeting customers’ requirements, this is not preferable.

[0016] Overcoming some of these bottlenecks e.g. masking in case of different off-flavor with aroma additives are possible, but not favorable due to elongated ingredient lists. Thus, it is highly beneficial to achieve the desired properties of the protein isolate during isolation and without adding additional substances to the final product. Such additional additives are not preferred in terms of achieving a clear label in the final product and fulfilling customer’s expectations towards an alternative food.

[0017] It was thus a major object of the present invention to provide methods to be in a position to modify the pea protein ingredient in a targeted way to preferably provide protein isolates having improved physicochemical properties in comparison to such products from a knownmethod for the production of protein isolates from pea, particularly having a better taste and an improved nutritional score.

[0018] Brief Description of the Drawings

[0019] Figure 1: Depiction of the results of the solubility test. A pea protein isolate manufactured according to the present invention with citric acid, acetic acid or lactic acid was compared to a method known from the state of the art including the treatment with hydrochloric acid. Different time points between 15 minutes and 120 minutes were tested.

[0020] Figure 2: Results of the foaming capacity tests as described in Example 4.

[0021] Definitions

[0022] The term “final application” as used herein refers to the final use of the pea protein isolate including the pea protein ingredient. Such a “final application” can be an alternative food product I composition as defined below.

[0023] An “alternative food” or “alternative nutrition” or “alternative (food) product I composition” as used herein refers to a food, including liquid food like beverages, which is usually a plant- or microorganism-based food that is an alternative to animal-derived food, including meat or dairy. Alternative food products may be of particular interest as an alternative source of proteins, but the term alternative food refers to any kind of nutritional building block, including proteins, carbohydrates, lipids, vitamins, minerals, fibers and the like that are suitable for food production and that are well accepted or even healthy as food and feed for human beings or farm animals and pets. An alternative food product or alternative food thus represents an “alternative consumable product” or an ingredient thereof. The ingredients of the present invention are useful to be implemented into an alternative food, an alternative feed or an alternative cosmetic. Thus, in particular, the term “alternative” means plant-based, i.e. providing an alternative to animal-based ingredients or products. An “alternative food product” as used herein may be present in liquid form, in semi-liquid form or in solid form. “Liquid” food products are e.g. any drinkable products including any beverages or soups, broths and sauces. “Semi-liquid” food products are e.g. yogurts, gelled products like desserts or pastes, spreads and dips. “Solid” food products are e.g. any products that require to be chewed when consumed. As used herein, an “alternative food product” refers to a product for human or animal consumption that is usually made with ingredients from animal sources, but in which the animal-sourced ingredient has been partially or fully replaced with a plant-based substitute ingredient. Non-limiting examples of alternative food products include alternative beverages such as a milk substitute or adrinkable yogurt substitute, or alternative food products such as an egg, beef, dairy, poultry, or seafood substitute. Other alternative food products include pet or animal feed products in which some or all of the animal-sourced ingredients are substituted with plant-based ingredients. The replacement or substitution of the animal-sourced ingredient may in some embodiments be e.g. more than 70%, more than 80%, more than 90%, or more than 95%. In other embodiments, the replacement or substitution of the animal-sourced ingredient may be 70% or less, such as 60%, 50%, 40%, etc. Alternative food products may also include non-dairy beverages such as sports drinks or smoothies. Alternative food products may further referto alternative nutritional products, such as plant-based powder, to be used as dietary or nutritional supplements.

[0024] An “alternative product” or an “alternative composition” as used herein, and any pea protein ingredient suitable for the production thereof of the present invention is specifically processed (industrially and / or mechanically and / or chemically and / or enzymatically) and a pea protein ingredient of this invention will usually be processed, isolated, concentrated and / or otherwise treated for inclusion in an alternative product or composition. Additionally, a pea protein ingredient of this invention will usually represent an intermediate ingredient, that was or that can be isolated from a plant representing one part or fraction of a final product or composition, or of a mixture or hybrid product. In an alternative product or composition, the pea protein ingredient of this invention as alternative part or fraction of the alternative product or composition thus substitutes a part or fraction that would be present in a commonly known and commonly fabricated product or composition, preferably, wherein it substitutes a part or fraction of animal or non-plant origin in the corresponding commonly known and commonly fabricated product.

[0025] Whenever the terms “composition” or “pea protein composition” is used herein, it refers to a composition of any of the protein ingredients, flours, concentrates or isolates disclosed herein that are used in an extracted form together with other ingredients from different origins to provide said composition. An alternative food or a cosmetic is thus also a composition in this sense. In this context, a “mixture” or “pea protein mixture” is a specific form of a composition, wherein pea protein fractions from different peas or even from different plants or other sources are mixed with each other to provide a basic protein mixture (and optionally further additives or ingredients of different nature and / or origin) comprising pea protein of the present invention.

[0026] A “consumable product” as used herein refers to goods that are usually understood to be used up or depleted during normal business operations, such as food and beverage. In line with the general understanding, there are two main types of consumables: durableconsumables, which are expected to last over a long period of time, and non-durable consumables, which are expected to be used up relatively quickly. Examples of consumables include perishable foods and beverages, paper products, ink cartridges, cleaning chemicals, gloves, and syringes. The consumable products particularly dealt with herein are non-durable consumables that are non-toxic when swallowed or applied on the human or animal body based on their intended use that are usually made of at least one organic raw materials (and optionally others), including food, beverages, gels, ointments, tooth paste and the like. Consumable products are, in particular, food products or cosmetic products, i.e. they are either ingested or applied to the body, e.g. the skin or hair, and thus used up or consumed.

[0027] As used herein, “dairy substitute” or “dairy substitute composition” or “dairy alternative” or “dairy alternative product” refer to compositions that mimic the general appearance, nutritional content, and / or taste of dairy products produced using animal milk products without containing animal-based milk or being substantially free of animal-based products, and includes hybrid products made with lab-grown, fermented and animal-based components such as protein components. The dairy substitute may be completely free of any animal-based milk or animal-based milk protein or almost free of any animal-based milk protein, such as e.g. 90% free, or 95% free of any animal-based milk protein. The dairy substitute may be a dairy-free cheese, a dairy-free yogurt, a dairy-free ice cream, and the like.

[0028] Notably, the term “food” as used herein refers to a food intended for human nutrition, whereas a “feed” as used herein refers to a feed.

[0029] As used herein “incubating” means a method step where a sample is maintained under specific conditions (such as temperature, time, and pH) to facilitate a desired biochemical reaction or interaction. In terms of the disclosure mentioned herein, “incubating” means maintaining a sample at a specific state for a specific time.

[0030] As used herein, “meat substitute” or “meat substitute composition” or “meat alternative” refers to compositions that mimic the general, organoleptic, and / or nutritional properties of consumable products produced using any type of meat or meat analog, including meat, fish, poultry, lab-grown and fermented meat products. This definition includes hybrid products made with lab-grown, fermented and animal-based components, such as protein components. A similar definition is used herein for “egg substitute”, “egg substitute composition,” and “egg alternative”.A “hybrid composition”, a “hybrid product” or a “hybrid alternative (consumable, including food / cosmetic etc.) composition or product” as used herein refers to an alternative product that at least partially comprises an “alternative food” or “alternative nutrition” or “alternative (food) product” comprising a plant-based ingredient according to the present invention, but which may comprise further ingredients.

[0031] The isoelectric point, also abbreviated as pl or IEP, is that pH at which a molecule, for instance a protein or a protein mixture, carries no net electrical charge, or is electrically neutral in the statistical mean, which is usually measured in solution for proteins. As it is known to the skilled person, the net charge on the molecule is affected by pH of its surrounding environment and can become more positively or negatively charged due to the gain or loss, respectively, of protons (H+).

[0032] An “organic acid” in terms of the present invention means a type of acidic compound that contains carbon and is typically derived from living organisms. It commonly features a carboxyl (-COOH) functional group, though other acidic groups like sulfonic (-SO3H) or phenolic (-OH) can also contribute to acidity. Organic acids are generally weak acids, meaning they do not fully dissociate in water. Examples include acetic acid (found in vinegar), citric acid (in citrus fruits), and lactic acid (in fermented foods). These acids play vital roles in biological processes, food preservation, and industrial applications.

[0033] The term “physicochemical properties” as used herein refers to all physical and chemical characteristics that influence the functionality, stability, and suitability of a pea protein ingredient for various applications. These properties include solubility, which affects dispersion in liquids; thermal stability, which influences denaturation and processing conditions; and water-holding, emulsifying, and foaming capacities and gelling capacities, which are critical for food and industrial applications. The term also describe the taste of the pea protein ingredient in the final application as well as the color of it.

[0034] A “(pea) protein flour” as used herein refers to an ingredient that contains milled peas, which are preferably dehulled before milling.

[0035] The terms Pisum sativum (L.) plant and pea plant, and short only pea, are used interchangeably herein, wherein the term pea is used in the context of the plant as a whole, but also to denote parts thereof, particularly seeds / fruits within pea pods.

[0036] A “pea protein ingredient” as used herein is to be understood as the total amount of protein that can be extracted from a pea fruit or seed (dry or fresh).The term “protein concentrate” is a protein ingredient with a concentration of about 30% to 60%. A “protein isolate” is an even more concentrated protein ingredient with a concentration of about 60% to about 100%. A “protein flour” represent the protein that can be obtained directly after dehulling and milling. As this protein flour is not yet heavily processed, it reflects the original content of protein ingredients rather directly. A “protein powder” is usually composed of fine, dry particles including the protein and the starch fraction of the pea protein isolate.

[0037] A ’’native” state of a protein is used herein to refer to a state, in which the protein is properly folded and / or assembled and therefore operative and functional. In contrast, in the denatured state, secondary through quaternary structure may be lost, retaining only the biomolecule's primary structure.

[0038] Description of the Invention

[0039] The present invention is directed to improvement of the physiochemical properties of a pea protein isolate making the isolate highly attractive for alternative food, animal nutrition, feed and cosmetics production, especially for products and compositions, which have a special requirement profile in terms of their properties.

[0040] In one aspect, there is thus provided a method of producing a pea protein isolate having improved physicochemical properties of a Pisum sativum plant, the method comprising the following steps:

[0041] i. providing at least one pea flour;

[0042] ii. isolating the protein ingredient from the pea flour involving the steps of: a) mixing the provided pea flour from step i. with at least one buffer and adjusting the pH of the solution to a range from about 6.5 to 9.5;

[0043] b) separating the soluble fraction and the solid fraction of the mixture obtained in step a. and obtaining a soluble and a solid fraction;

[0044] c) providing at least one organic acid individually being selected from lactic acid, citric acid and acetic acid, or a combination thereof, to the soluble fraction; d) adjusting the pH to the isoelectric point of the pea protein ingredient, preferably in a range from about 4.0 to 5.0, and obtaining a soluble and a solid fraction, wherein the solid fraction contains the pea protein ingredient;

[0045] Hi. optionally drying the obtained solid fraction and obtaining a pea protein isolate.Preferably, the pea protein ingredient is not heated to a temperature of 60 °C or higher, preferably 50 °C or higher, most preferably 40 °C or higher during step ii and / or before or during step Hi.

[0046] Step i. of the method according to the present invention may include providing a commercially available pea protein flour or manufacturing a flour by dehulling and milling of the dried peas.

[0047] Step ii. a) involves the adjustment of the pH of the solution after adding at least one buffer. The pH to which the mixture comprising buffer and the at least one pea flour from step i. is adjusted ranges around a pH of 8.0 ± 1.5. Step ii. a) leads to the formation of a soluble fraction and a solid fraction within the mixture.

[0048] In step ii. b), the soluble and the solid fraction of the mixture of step ii. a) are separated. The soluble fraction contains the protein ingredient and the solid fraction comprises the solid components of the protein flour, in particular starch and fibers. The soluble fraction is then used for further processing.

[0049] In the subsequent step ii. c), at least one organic acid is added to the soluble fraction obtained in step ii. b). The organic acids used in this step are all approved to be used in an organic process and it was surprisingly found that they have a beneficial influence on the physicochemical properties of the obtained pea protein isolate. Such beneficial properties can be taste, protein composition, protein nativity, emulsion stability in the final application, the solubility, gelling property, foaming property, thermal stability or the color of the protein isolate. It was especially observed that the solubility of the obtained pea protein isolate is higher using a method according to the present invention than using a method known from the state of the art. Furthermore, as the use of hydrochloric acid is a standard procedure known from the state of the art, this leads during processing to a high sodium chloride deposition in the final protein isolate and thus to a salty taste, which is not preferable. As initially described, a high sodium chloride content is not preferable for the manufacturing of alternative food products. It was surprisingly observed that the use of organic acids, which have a weaker dissociation profile in comparison to hydrochloric acid, could be used observing the same process yield and only minor or no losses of pea protein ingredient in the process according to the present invention.

[0050] Also, a combination of one, two or all of the organic acids citric acid, acetic acid and lactic acid can be used. A combination of citric acid and acetic acid, acetic acid and lactic acid or citric acid and lactic acid may be used. With regard to the concentration of the acids, it ispreferred that the concentration of the combined acid is equivalent to the concentration of a single acid.

[0051] It was especially observed that adding or incubating the soluble fraction comprising the protein ingredient leads to an improved taste and solubility of the protein isolate in the final product in contrast to treatments as described and available for food grade production in the relevant technical field.

[0052] It was found that it has a significant impact on the physicochemical properties of the obtained pea protein isolate, which acid is used in step ii. c) of the method according to the invention. This is surprising, because it would be expected that, at the same pH, the same properties would be observed for the product, irrespective of which acid was used to adjust the pH and precipitate the protein.

[0053] The properties obtained with the different acids are compared in Table 1.

[0054] Table 1 : Properties obtained with different acids

[0055] acid conductivity solubility G*(gelling capacity)

[0056] HCI low high medium-high (1030)

[0057] lactic acid medium high medium (917)

[0058] citric acid medium-high low very high (1585)

[0059] acetic acid high medium low (606)

[0060]

[0061] Specifically, it was found that, when lactic acid is provided in step ii. c) of the method, the resulting pea protein isolate has a very high solubility compared to other acids used. This is demonstrated in example 6.

[0062] In one embodiment of the method described above, lactic acid is provided as the organic acid in step ii c).

[0063] When lactic acid is used in step ii c) of the method according to the invention, the resulting pea protein isolate has a high solubility, in particular at least 50 wt.-%, preferably at least 60 wt.-%, more preferably at least 65 wt.-%, most preferably at least 70 wt.-%. The resulting pea protein isolate may comprise at least 500 mg soluble protein per g protein isolate sample, preferably at least 600 mg soluble protein per g protein isolate sample, most preferably at least 700 mg soluble protein per g protein isolate sample. The solubility ispreferably given in aqueous compositions, in particular in food products or beverages such as juice, alternative milks or creams and sauces and preferably determined at 25 °C. It was also found that, when citric acid is provided in step ii c) of the method, the resulting pea protein isolate has a very high gelling capacity and therefore can form gels with a particularly high gel strength. This is demonstrated in example 5.

[0064] In one embodiment of the method described above, citric acid is provided as the organic acid in step ii. c).

[0065] When citric acid is used in step ii. c) of the method according to the invention, the resulting pea protein isolate has a high gelling capacity and / or forms gels with a high gel strength, in particular with a complex shear modulus of at least 1000 Pa, preferably at least 1200 Pa. It was also found that, when acetic acid is provided in step ii. c) of the method, the resulting pea protein isolate has a low gelling capacity as demonstrated in example 5 and / or a high conductivity.

[0066] In one embodiment of the method described above, acetic acid is provided as the organic acid in step ii. c).

[0067] When acetic is used in step ii. c) of the method according to the invention, the resulting pea protein isolate has a low gelling capacity or forms gels of low strength, in particular with a complex shear modulus below 700 Pa, preferably below 600 Pa, and / or a high conductivity, in particular at least 1500 microSiemens / cm, preferably at least 1700 microSiemens / cm. Thus, by using the respective acids in step ii. c) of the method according to the invention, the properties of the resulting pea protein isolate can be adjusted and fine-tuned for specific applications. For example, lactic acid can be used when high solubility is required but no particularly strong gelling capacity is desired. This may be suitable for the production of drinkable products. If gelled structures are the final application, citric acid can be used advantageously and if pumpable and low viscosity applications are intended, acetic acid would be suitable for use in step ii. c).

[0068] In step ii. d), the pH of the soluble fraction is adjusted to the isoelectric point of the pea protein ingredient, wherein the pea protein ingredient is then precipitated and contained in the resulting solid fraction. This step as well as step ii. b) could also include at least one washing step of the pea protein ingredient. Furthermore, adjusting of the pH may be done optionally with at least one organic acid differing from the organic acid used in step ii. c).Furthermore, the at least one washing step could be done after concentrating the precipitated pea protein fraction and before subjecting the fraction containing the pea protein to an optional drying step.

[0069] The isoelectric point of a molecule or a mixture of molecules can be determined by a suitable method known by the person skilled in the art. Such a suitable method can be e.g. the titration and parallel zeta potential measurement. The pH value of the solution at which the zeta potential is zero corresponds to the isoelectric point of the molecule or the mixture. In one embodiment of the method according to the present invention, the organic acid provided in step ii. c) is provided in a concentration of at least 40 % vol. / vol., at least 45 % vol. / vol., at least 50 % vol. / vol., at least 55 % vol. / vol., at least 60 % vol. / vol., at least 65 % vol. / vol., at least 70 % vol. / vol., at least 75 % vol. / vol., at least 80 % vol. / vol., at least 85 % vol. / vol., at least 90% vol. / vol., or at least 95% vol. / vol., preferably at 60 % vol. / vol. to 90 % vol. / vol. and especially preferably 75 % vol. / vol. to 85% vol. / vol..

[0070] A 80 % vol. / vol. concentrated acid may describe a solution containing 80 % organic acids and usually 20 % water. The amount of acid compound in such a solution determines the overall acidity of the solution. In terms of the present invention, it is preferred that the solution contains at least 60 % vol. / vol. acids.

[0071] In the method according to the invention, the pea protein ingredient is preferably not subjected to any heat treatments during the production of the pea protein isolate. In particular, it is not heated to 60 °C or higher, preferably 50 °C or higher, more preferably 40 °C or higher.

[0072] Advantageously, by avoiding heat treatments, the pea protein ingredient is maintained in a native or mostly native state. This ensures a high solubility and retains the ability to form an even gel network. On the other hand, when the pea protein ingredient is denatured, gelling is more paste-like and aggregates tend to hold water, resulting in an uneven gel formation.

[0073] Preferably, the pea protein isolate obtained by the method of the present invention, is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% at least 90% in native form. In addition, preferably the pea protein isolate obtained by the method of the present invention is not coagulated.In yet another embodiment, there is provided a method, wherein the at least one buffer in step ii. a) is selected from water, ethanol, a solution of phosphates and its salts or mixtures thereof.

[0074] In terms of the present invention, the use of water as buffer is particularly preferred. In one embodiment of the methods as described herein, the soluble and the solid fraction are separated in step ii. b) with a technique selected from the group consisting of sedimentation, centrifugation and / or filtration.

[0075] In terms of the present invention, centrifugation and / or sedimentation is particularly preferred.

[0076] Yet another embodiment of the present invention relates to a method, wherein step ii. c) includes an incubation after addition of the at least one organic acid, preferably while stirring the mixture.

[0077] Incubation of the soluble fraction including the pea protein ingredient with the at least one organic acid leads to the precipitation of the protein in a physicochemical state, which has desired and / or improved properties as described herein in comparison to the treatment with hydrochloric acid as known from the state of the art.

[0078] In one embodiment of the present invention, step ii. d) includes a step of incubation of the mixture, preferably under stirring for a time of 1 to 90 minutes, preferably 1 to 60 minutes and especially preferably 1 to 30 minutes.

[0079] In yet another embodiment of the present invention, the drying in step Hi. is done via spraydrying, freeze drying or thermal drying.

[0080] In terms of the present invention, it is particularly preferred that the drying is done via spraydrying.

[0081] A second aspect of the present invention relates to a pea protein isolate, preferably having at least one altered physicochemical property compared to a pea protein isolate of a Pisum sativum plant not obtained with a method according to the present invention, obtained or obtainable by a method according to the invention.

[0082] Such physicochemical properties may be selected according to one embodiment of the second aspect of the present invention from aste, solubility, emulsion stability, color of thepea protein isolate, gelling capacity, foaming capacity, thermal stability, composition of proteins and profile of the composition of volatile compounds.

[0083] In one embodiment, the at least one altered physicochemical property is selected from the group consisting of taste, solubility, emulsion stability, color, gelling capacity, foaming capacity, thermal stability, protein composition and profile of the composition of volatile compounds. It was especially observed that the pea protein isolate obtained or obtainable by a method according to the present invention has a more acceptable taste in comparison to a pea protein isolate that is obtained by a method known from the state of the art. Furthermore it shows an increased solubility of the pea protein ingredient, which is also shown in Figure 1.

[0084] As mentioned above, it was surprisingly found that it has a significant impact on the physicochemical properties of the obtained pea protein isolate, which acid is used in step ii. c) of the method according to the invention, even though it would be expected that, at the same pH, the same properties would be observed for the product, irrespective of which acid is used.

[0085] Therefore, preferably the at least one physicochemical property is altered with respect to a pea protein isolate obtained by a method using an inorganic acid, in particular HCI to precipitate the pea protein ingredient.

[0086] In one embodiment, the pea protein isolate is obtained by a method, wherein lactic acid is used in step ii. c) and the at least one altered physicochemical property is solubility and is altered, in particular increased, with respect to a pea protein isolate obtained by a method using an inorganic acid and / or an organic acid other than lactic acid, in particular citric acid or acetic acid, to precipitate the pea protein ingredient.

[0087] In one embodiment, the pea protein isolate is obtained by a method, wherein citric acid is used in step ii. c) and the at least one altered physicochemical property is gelling capacity and / or gel strength and is altered, in particular increased, with respect to a pea protein isolate obtained by a method using an inorganic acid and / or an organic acid other than lactic acid, in particular lactic acid or acetic acid, to precipitate the pea protein ingredient. In one embodiment, the pea protein isolate is obtained by a method, wherein acetic acid is used in step ii c) and the at least one altered physicochemical property is gelling capacity and / or gel strength and is altered, in particular decreased, with respect to a pea protein isolate obtained by a method using an inorganic acid and / or an organic acid other than lactic acid, in particular lactic acid or citric acid, to precipitate the pea protein ingredient.Specifically, it was found that, when lactic acid is provided in step ii. c) of the method, the resulting pea protein isolate has a very high solubility compared to other acids used. This is demonstrated in example 6. Moreover, it was found that, when citric acid is provided in step ii. c) of the method, the resulting pea protein isolate has a very high gelling capacity and can form gels with a particularly high gel strength. This is demonstrated in example 5. Finally, it was also found that, when acetic acid is provided in step ii. c), the resulting pea protein isolate has a low gelling capacity as demonstrated in example 5 and / or a high conductivity.

[0088] Therefore, in a preferred embodiment of the pea protein isolate according to the invention, the pea protein isolate has a higher solubility when lactic acid is provided in step ii. c) or the pea protein isolate has higher gel strength when citric acid is provided in step ii. c), or the pea protein has lower gelling capacity when acetic acid is provided in step ii. c).

[0089] In particular, the present invention relates to a pea protein isolate obtained or obtainable by a method according to the invention as described in any of the embodiments mentioned above, wherein lactic acid is provided as the organic acid in step ii. c).

[0090] In one embodiment, the pea protein isolate obtained or obtainable by a method according to the invention as described in any of the embodiments mentioned above, wherein lactic acid is provided as the organic acid in step ii c), has a high solubility, in particular at least 50 wt.-%, preferably at least 60 wt.-%, more preferably at least 65 wt.-%, most preferably at least 70 wt.-%. The pea protein isolate may comprise at least 500 mg soluble protein per g protein isolate sample, preferably at least 600 mg soluble protein per g protein isolate sample, most preferably at least 700 mg soluble protein per g protein isolate sample. The solubility is preferably given in aqueous compositions, in particular in food products or beverages such as juice, alternative milks or creams and sauces and determined at 25 °C. In particular, the present invention relates to a pea protein isolate obtained or obtainable by a method according to the invention as described in any of the embodiments mentioned above, wherein citric acid is provided as the organic acid in step ii. c).

[0091] In one embodiment, the pea protein isolate obtained or obtainable by a method according to the invention as described in any of the embodiments mentioned above, wherein citric acid is provided as the organic acid in step ii. c), has a high gelling capacity and / or forms gels with a high gel strength, in particular with a complex shear modulus of at least 1000 Pa, preferably at least 1200 Pa.In particular, the present invention relates to a pea protein isolate obtained or obtainable by a method according to the invention as described in any of the embodiments mentioned above, wherein acetic acid is provided as the organic acid in step ii c).

[0092] In one embodiment, the pea protein isolate obtained or obtainable by a method according to the invention as described in any of the embodiments mentioned above, wherein acetic acid is provided as the organic acid in step c) has a low gelling capacity or forms gels of low strength, in particular with a complex shear modulus below 700 Pa, preferably below 600 Pa, and / or has a high conductivity, in particular at least 1500 microSiemens / cm, preferably at least 1700 microSiemens / cm.

[0093] In the method according to the invention, by preferably avoiding any heat treatments, in particular temperatures of 60 °C or higher, 50 °C or higher or 40 °C or higher, the pea protein ingredient is maintained in a native or mostly native state. This ensures a high solubility and retains the ability to form an even gel network. On the other hand, when the pea protein ingredient is denatured, gelling is more paste-like and aggregates tend to hold water, resulting in an uneven gel formation.

[0094] In any of the embodiments of the pea protein isolate described above, the pea protein isolate is preferably at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% at least 90% in native form. In addition, preferably the pea protein isolate is not coagulated.

[0095] A third aspect of the present invention relates to a mixture comprising a pea protein isolate of a Pisum sativum plant as described above and at least one further additive and / or ingredient.

[0096] The skilled person being a food chemist, expert, a chemist, or a process engineer will well be in a position to adjust the final content of a product to achieve the optimum conditions guaranteeing easy and efficient manufacturing as well as a hygienically safe, food- or cosmetics-grade product with the right degree of stability and non-perishable nature as needed.

[0097] A fourth aspect of the present invention relates to an alternative consumable product, or a hybrid composition or product, comprising at least one pea protein isolate of a Pisum sativum plant according to the invention, or comprising at least one mixture according to the invention, preferably wherein the alternative consumable product has an improved color in comparison to a reference material.Such an alternative consumable product may be a meat substitute product or a dairy alternative product. The meat products may include but are not limited to ground meat analogues, shredded meat analogues, and cut or fileted meat analogues. The dairy products may be hard, semi-hard, semi-soft, or soft cheese products, or spoonable or pourable dairy products, also including yogurt, ice cream and the like.

[0098] In one embodiment, the alternative consumable product, or the hybrid composition or product is a drinkable alternative consumable product, in particular a shake or an ultra-heat treated drink, preferably a plant based milk analog, and the pea protein isolate is obtained or obtainable by a method according to any one of the embodiments described above, wherein lactic acid is provided in step ii. c).

[0099] In one embodiment, the alternative consumable product, or the hybrid composition or product is a gelled alterative consumable product, in particular a plant based yogurt or meat analog or dessert, and the pea protein isolate is obtained or obtainable by a method according to any one of the embodiments described above, wherein citric acid is provided in step ii. c).

[0100] In one embodiment, the alternative consumable product, or the hybrid composition or product is a pumpable or low viscosity alternative consumable product, and the pea protein isolate is obtained or obtainable by a method according to any one of the embodiments described above, wherein acetic acid is provided in step ii. c).

[0101] A reference material can be all alternative consumables products, hybrid compositions or products that contain a pea protein isolate, which is not obtained according to the present invention. In particular, obtained by a method using an inorganic acid, in particular HCI to precipitate the pea protein ingredient.

[0102] In certain embodiments, the pea protein isolate, the pea protein texturate, the pea protein powder, or a pea protein flake of a Pisum sativum plant is incorporated into a milk alternative product or a meat alternative product.

[0103] According to certain embodiments disclosed herein, the alternative consumable product is a hybrid product that comprises a pea protein isolate of the present invention as well as at least one further additive and / or ingredient. Besides additives and ingredients typically used for food and cosmetic production, also at least one lab-grown material can be used. A lab-grown material as used herein refers to a cultured and / or fermented cell, and the material (any biomass, cellular and non-cellular, including supernatant) obtained therefrom, including bacterial cells, fungal cells, including filamentous fungi, particularly fungi and fungibiomass suitable as meat substitute, and cultured animal cells, including hepatocytes, myoblasts, osteoblasts, fibroblasts, lipoblasts, odontoblasts, adult neuronal progenitor cells, neural stem cells, multipotent stem cells from subventricular forebrain region, ependymal-derived neural stem cells, hematopoietic stem cells, liver-derived hematopoietic stem, marrow-derived stem cell, adipo-fibroblasts, adipose-derived stem cells, islet- cells producing stem cells, pancreatic-derived pluripotent islet-producing stem cells, mesenchymal stem cells, placenta cells, bone marrow stromal cells, muscle side population cells, bone marrow-derived recycling cells, blood-derived mesenchymal precursor cells, bone- marrow derived side population cells, muscle precursor cells, circulating skeleton stem cells, neural progenitor cells, multipotent adult progenitor cells, mesodermal progenitor cells, spinal cord progenitor cells and spore-like cell, and any combinations thereof, wherein the cell is not derived from a human embryo.

[0104] In certain embodiments, the consumable product, preferably an alternative food product or a hybrid product, as described herein may include one or more lipid composition(s), for example a fat, an oil, or combinations thereof. In general, fats refer to lipid compositions that are solid at room temperature, whereas oils are liquid at room temperature. The lipid compositions may include saturated fatty acids (also referred to as “saturated fats”), unsaturated fatty acids (also referred to as “unsaturated fats”), or combinations thereof. The lipid composition may include, but are not limited to, vegetable oil, coconut oil, palm oil, sunflower oil, soy oil, canola oil, or combinations thereof. The consumable product may include between 1% and 80%, between 1% and 70%, between 1% and 10%, between 1% and 5%, between 5% and 30%, between 10% and 25%, between 10% and 75%, or between 15% and 70% by weight of a lipid composition depending on the type of dairy substitute. An ordinarily skilled artisan will understand the appropriate lipid composition inclusion rate for a given composition.

[0105] The alternative food product, or the alternative cosmetic product, or a hybrid product, may include water as needed. For example, the product may include between 1% and 80%, between 5% and 75%, between 15% and 70%, between 45% and 65%, between 50% and 60%, between 1% and 20%, or between 5% and 15% by weight of water depending on the type of consumable product.

[0106] In some embodiments, the consumable product comprising a pea protein isolate of a Pisum sativum plant, having improved physicochemical properties includes both water and a plant-based ingredient. The total of the water and plant-based ingredient may be e.g. between 50% and 95% or between 60% and 90% by weight of the composition.In certain embodiments, the consumable product comprising a pea protein ingredient, pea protein isolate of a Pisum sativum plant having improved physicochemical properties may include fiber. The fiber may include, but is not limited to, pectin, apple fiber, psyllium, flax fiber, rice bran extract, Konjac flour, and the like. The consumable product may include between 0.01% (wt) and 3% (wt), between 0.05% (wt) and 2% (wt), or between 0.1% (wt) and 2% (wt) of fiber. The consumable product may include fiber in an amount up to 0.5% (wt), up to 1% (wt), up to 1.5% (wt), up to 2% (wt), up to 2.5% (wt), or up to 3% (wt). In certain embodiments, the consumable product comprising a pea protein ingredient, protein isolate of a Pisum sativum plant having improved physicochemical properties may include starch. The starch may include a pregelatinized starch, a modified starch, or combinations thereof. The starch may include, but is not limited to, com starch, potato starch, tapioca starch, and the like. The consumable product may include between 0.5% (wt) and 25% (wt), between 1.0% (wt) and 20% (wt), or between 2% (wt) and 18% (wt) of starch. The consumable product may include a hydrocolloid. For example, the consumable product may include guar gum, xanthan gum, locust bean gum, carrageenan, cellulose, konjac gum, and combinations thereof. The consumable product may include between 0.01% and 5%, between 0.05% and 4.5%, between 0.1% and 4.0%, or between 0.5% and 3.8% by weight of hydrocolloid. The consumable product may include up to 5%, up to 4.5%, up to 4.0%, up to 3.8%, up to 3.5%, up to 2.5%, up to 2.0%, or up to 1.0% by weight of hydrocolloid.

[0107] In certain embodiments, the consumable product comprising a pea protein ingredient or protein isolate of a Pisum sativum plant having improved physicochemical properties may include lecithin. The consumable product may include between 0.01% and 10%, between 0.05% and 8.0%, or between 0.1% and 5% by weight lecithin.

[0108] In certain embodiments, the consumable product comprising a pea protein ingredient or protein isolate of a Pisum sativum plant having improved physicochemical properties may include a preservative. For example, the consumable product may include a preservative such as, but not limited to potassium sorbate. The consumable product may include a preservative in an amount up to 0.1%, up to 0.5%, or up to 1.0% by weight of the consumable product. The consumable product may include a flavor or seasoning. For example, the consumable product may include a natural or artificial flavor(s) and / or seasonings. Seasonings may include, but are not limited to, sweetener(s), salt (e.g., sodium chloride, potassium chloride, and the like), cocoa, chocolate, cinnamon, nutmeg, coconut, almond, combinations thereof, and the like. The consumable product may include between 1% and 20%, between 1.5% and 10%, between 5% and 20%, or between 2% and18% of a flavor or seasoning. The consumable product may be free of any flavors or seasoning. In some embodiments, the consumable product may include between 0.001% and 3.0%, between 0.01% and 2.0%, or between 0.025% and 1.75% of a salt. The consumable product may be free of salt.

[0109] In certain embodiments, the consumable product comprising a pea protein ingredient or protein isolate of a Pisum sativum plant having improved physicochemical properties may include a sweetener. Suitable sweeteners are known and described in the art. The sweetener can be at least one of a non-caloric sweetener or a caloric sweetener. The sweetener can be any type of sweetener, for example, a sweetener obtained from a plant or plant product, or a physically or chemically modified sweetener obtained from a plant, or a synthetic sweetener. Exemplary sweeteners include steviol glycosides, mogrosides, sucrose, fructose, glucose, erythritol, maltitol, lactitol, sorbitol, mannitol, xylitol, tagatose, trehalose, galactose, rhamnose, cyclodextrin (e.g., a-cyclodextrin, b-cyclodextrin, and g-cyclodextrin), ribulose, threose, arabinose, xylose, lyxose, allose, altrose, mannose, idose, lactose, maltose, invert sugar, isotrehalose, neotrehalose, palatinose or isomaltulose, erythrose, deoxyribose, gulose, idose, talose, erythrulose, xylulose, psicose, furanose, cellobiose, glucosamine, mannosamine, fucose, fuculose, glucuronic acid, gluconic acid, glucono-lactone, abequose, galactosamine, xylo-obgosaccharides (xylotriose, xylobiose and the like), gentio- obgoscaccharides (gentiobiose, gentiotriose, gentiotetraose and the like), galacto- obgosaccharides, sorbose, ketotriose (dehydroxyacetone), aldotriose (glyceraldehyde), nigero- obgosaccharides, fructoobgosaccharides (kestose, nystose and the like), maltotetraose, maltotriol, tetrasaccharides, mannan-oligosaccharides, maltooligosaccharides (maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose and the like), dextrins, lactulose, melibiose, raffmose, rhamnose, ribose, sucralose, acesulfame K, aspartame, saccharin, coupling sugars, soybean oligosaccharides, and combinations thereof. D- or L-configurations can be used when applicable.

[0110] In certain embodiments, the consumable product comprising a pea protein ingredient, protein isolate of a Pisum sativum plant having improved physicochemical properties may include an acid. Suitable acids include, but are not limited to, citric acid, lactic acid, sorbic acid, malic acid, combinations thereof, and the like. The consumable product may include an acid in an amount up to 0.001%, up to 0.005%, up to 0.01%, up to 0.1%, up to 1.0%, up to 1.5%, or up to 2.0% of the consumable product. The consumable product may include between 0.0001% and 2.0%, between .0002% and 1.5%, between 0.0003% and 1.0% by weight of an acid.In certain embodiments, the hybrid composition or product may comprise at least one portion being a pea protein ingredient or protein isolate of a Pisum sativum plant of the present invention and at least one portion being a lab-grown material, preferably wherein the lab-grown material stems from a bacterial fermentation, or from a fungus, preferably from a filamentous fungus. Filamentous fungal biomass, usually named mycoproteins, is a suitable meat substitute since it is nutritious and has filaments and thus a texture perfectly mimicking meat fibrils. Regarding the type of residual water, nutrient supplementation, optimum conditions for biomass production, and characteristics of the mycoproteins, the optimum growth condition can be at about pH of 4.5. Mycoprotein usually contains 19.44% (wt. / wt.) protein with a high crude fiber content of 8.51% (wt. / wt.) and a low fat content of 1.56% (w / w). In addition, the amino acid and fatty acid contents are dominated by glutamic acid and polyunsaturated fatty acids, which are associated with an umami taste (Wikandari et al., 2023, https: / / doi.org / 10.3390 / molecules28030997). Particularly the physicochemical and the viscoelastic properties of mycoproteins and the pea protein ingredients of the present invention allow that both sources of alternative proteins are easily compounded and used together for food, particularly alternative meat or fish design, as the pl values of the major protein fractions and the viscoelastic properties are perfectly suitable to produce food with a high nutrition value and an excellent texture and stability.

[0111] In one embodiment of the fourth aspect, the alternative food or the cosmetic composition or product, or the hybrid composition or product thereof has a pH in the range of about 3.5 to about 8.5, preferably a pH from about 4.5 to about 8.

[0112] In a fifth aspect the present invention relates to a use of a protein isolate of a Pisum sativum plant as defined in terms of the present invention, ora use of a mixture as defined according to the present invention for preparing an alternative consumable product or hybrid composition or product thereof, preferably wherein the consumable product or hybrid composition or product is a food product or a cosmetic composition or product.

[0113] As explained above, by using the respective acids in step ii. c) of the method according to the invention, the properties of the resulting pea protein isolate can be adjusted and finetuned for specific applications. For example, lactic acid can be used when high solubility is required but no particularly strong gelling capacity is desired. This may be suitable for the production of drinkable products. If gelled structures are the final application, citric acid can be used advantageously and if pumpable and low viscosity applications are intended, acetic acid would be suitable in step ii. c).

[0114] Preferably, a protein isolate of a Pisum sativum plant is obtained or obtainable by a method according to any of the embodiments described above, wherein lactic acid is provided instep ii. c) and the pea protein isolate is used for preparing a drinkable alternative consumable product, in particular a shake or an ultra-heat treated drink, preferably a plant based milk analog, or a protein isolate of a Pisum sativum plant is obtained or obtainable by a method according to any of the embodiments described above, wherein citric acid is provided in step ii. c) and the pea protein isolate is used for preparing a gelled alternative consumable product, in particular a plant based yogurt or meat analog or dessert, or a protein isolate of a Pisum sativum plant is obtained or obtainable by a method according to any of the embodiments described above, wherein acetic acid is provided in step ii. c) and the pea protein isolate is used for preparing a pumpable or low viscosity alternative consumable product.

[0115] In one embodiment, the present invention relates to the use of a protein isolate of a Pisum sativum plant obtained or obtainable by a method according to the invention, wherein lactic acid is provided in step ii. c) for preparing a drinkable alternative consumable product, in particular a shake or an ultra-heat treated drink, preferably a plant based milk analog. In another embodiment, the present invention relates to the use of a protein isolate of a Pisum sativum plant obtained or obtainable by a method according to the invention, wherein citric acid is provided in step ii. c) for preparing a gelled alternative consumable product, in particular a plant based yogurt or meat analog or dessert.

[0116] In a further embodiment, the present invention relates to the use of a protein isolate of a Pisum sativum plant obtained or obtainable by a method according to the invention, wherein acetic acid is provided in step ii. c) for preparing a pumpable or low viscosity alternative consumable product. While several possible aspects are disclosed above, embodiments of the present invention are not so limited. These exemplary aspects are not intended to be exhaustive or to unnecessarily limit the scope of the invention, but instead were chosen and described in order to explain the principles of the present invention so that others skilled in the art may practice the invention. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0117] The present invention is, in particular, captured by any one or any combination of one or more of the above-mentioned aspects, with any other statement and / or embodiments. The following Examples serve to provide further details on embodiments and enablement of the invention, but do not necessarily limit the scope of the invention.Example 1

[0118] 1. Dehulling of peas and producing a pea flour

[0119] For the dehulling step, the whole peas are put into a dosing funnel of a dehuller. Using the dosing channel, the speed and amount of peas falling into the dehulling chamber can be adjusted. The slit between the dehuller discs can be adjusted to the size of the peas. The mix of dehulled whole and split peas and their hulls is collected in a drawer below the discs and is transferred into the dosing funnel of the airleg separator in order to sort them apart. The air flow to the different chambers therein can be adjusted with two screws. Peas that are still not dehulled, are transferred back to the dehuller while adjusting the slit accordingly. Dehulled peas are then milled with an ultracentrifugal mill and the mentioned components, carefully dosing them with a scoop. The resulting flour is collected in a bowl, and residues on parts of the mill are brushed off in order to clean them.

[0120] 2. Manufacturing of a pea protein isolate from pea flour according to the present invention

[0121] Material used:

[0122] Pea flour

[0123] - IM NaOH

[0124] 80% lactic acid, citric acid or acetic acid

[0125] Distilled water

[0126] 3L and 5L beakers

[0127] Lab scale PCE Instruments PCE-BSH 10000

[0128] Propeller stirrer VWR VOS 40 digital

[0129] Magnetic stirrer IKA RH 2 basic

[0130] Centrifuge Eppendorf 5910 Ri cooled, with 4 centrifuge bottles 1L Disperser / homogenizer IKA ultraturrax T50 digital with S 50 N G45G disperser tool

[0131] pH meter Mettler Toledo FiveEasy F20

[0132] Spoon, silicone spatula and metal spatulaSpray dryer Biichi S-300

[0133] Pea flour was dispersed into distilled water while stirring with a propeller stirrer at around 400 rpm in a ratio of S:L (Solid to Liquid) of 1 :7, e.g. 500 g flour and 3000 mL water. The pH value of the dispersion was measured and adjusted using a pH meter and 1M NaOH until it reached pH 8. Extraction was conducted at 150 rpm at room temperature for 1 h. The dispersion was then transferred into the centrifugation bottles and centrifuged at 4347 x g for 25 min to separate the dissolved protein-rich supernatant from the pellet rich in starch and fibers. The pellet was disposed. The supernatant was transferred to the 3L beaker and stirred with the magnetic stirrer in order at 500 rpm to carefully dose the 80% lactic acid, citric acid or acetic acid to precipitate the protein. The pH was adjusted to the isoelectric point of the proteins at 4.5, and the protein dispersion was stirred for 10 min to help the proteins agglomerate better. The dispersion was then centrifuged at 4347 xg for 25 min at 7 °C to lower the protein solubility further and improve the protein yield. The resulting supernatant was disposed, and the protein pellet was carefully removed from the bottles with spatulas and transferred into a beaker. The protein was washed with distilled water at a ratio of S:L of 1 :5, using a ultraturrax disperser. The washed protein was again centrifuged under aforementioned conditions, and collected in a beaker. Neutralisation was carried out using the ultraturrax homogeniser, distilled water, until a protein concentration of about 10 % was reached, and 1M NaOH. The neutralized protein solution was then spray-dried under the following conditions: inlet temperature 130 °C, spraying gas flow 900 L / h.

[0134] Example 2: Comparison of the method according to the invention and method known from the state of the art (sedimentation test)

[0135] The solubility of the pea protein isolate manufactured according to the present invention in comparison to a pea protein isolate manufactured according to the state of the art was examined.

[0136] The following material was used for the solubility tests:

[0137] 250 mL glass measuring cylinder

[0138] Silicone spatula extra long

[0139] Protein isolate manufactured according to example 1 (Protein isolate can be used as an equivalent for all protein extracts, isolates and the like, in the present experiment a protein isolate with a protein content of 85 % was used) Distilled waterCamera

[0140] Stopwatch

[0141] Sample preparation

[0142] For the preparation of samples, 150 ml of distilled water was meticulously measured using a graduated cylinder and subsequently transferred into beakers. To this, 7.5 g of protein isolate powder was added. The mixture was then stirred thoroughly until a completely homogeneous solution was achieved.

[0143] Sample treatment and measurement

[0144] For each sample, a stopwatch was provided, and the time was meticulously recorded. Photographs of the beakers were taken at intervals of 15 minutes, 30 minutes, 1 hour, and 2 hours. At the conclusion of the treatment, the pH value of each beaker was measured.

[0145] Sample analysis

[0146] The evaluation of the samples was conducted visually, assessing the sedimentation of the isolate and the visual homogeneity.

[0147] Results

[0148] The results of the experiments can be seen in Figure 1. The samples treated with citric acid, lactic acid, and acetic acid showed no precipitation even after 2 hours. In contrast, the reference sample from the process involving the use of HCI according to the state of the art exhibited significant sedimentation (as can be seen at the bottom of the beaker (inside the black square). This indicates that the solubility of proteins is optimized when using organic acids compared to hydrochloric acid (HCI).

[0149] Example 3: Measuring of pea protein isolate color

[0150] 1 g of pea protein isolate was placed in a Petri dish. The isolate was then compressed with the spatula to create an even surface. This even surface was necessary to ensure a reliable color measurement. Afterwards, the Videometerlab measured the reflection of the sample placed in the sample chamber at the following wavelengths: 365, 405, 470, 515, 540, 590, 645, 780, 850, 970, 430, 450, 490, 570, 630, 660, 690, 880 and 940 nm.

[0151] The reflection was examined using VideometerLab. Analysis of the obtained data was done using the VideometerLab software.Mesurement with the VideometerLab generates values from the reflection measurement raw data. In general, VideometerLab captures multispectral images at 18 different wavelengths ranging from 405 to 970 nm. The acquisition system, records the surface reflections with a standard monochrome charged coupled device chip (CCD). The sample is placed inside a sphere, called Ulbricht sphere, which has a matte white coating so as to ensure a diffused and spatially homogenous illumination of the sample. At the rim of the sphere, light emitting diodes (LEDs) with narrow-band spectral radiation distribution are positioned side by side. During data acquisition, the diodes are strobing successively, resulting in a monochrome image with 32-bit floating point precision for each wavelength. Finally, a data cube of spatial and spectral data for each sample of size m x n x 18 (where m x n is the image size in pixels) is acquired. The whole system, is developed in order to guarantee the reproducibility of the collected images, and so it can be used in comparative studies of time series studies, or across a large variety of different samples. The acquisition process results to a plethora of data, either representing samples in a time series experiments or samples under different conditions and / or treatment (e.g. packaging conditions). Each sample image cube contains spatial as well as spectral information. Spectral information for every pixel lies at the spectral axis, whereas spatial information (x-y plane) gives the reflectance value of the sample at the pixel’s location and at the specific wavelength. (Tsakanikas et al., 2015).

[0152] The following measurements were performed with the VideometerLab as described above. The CIELAB values L*, a*, b* as well as the H_value and Stensby Index were calculated from the measurements. The results are shown in Table 2.

[0153] Table 2: Color measurements

[0154] Acid a' b* Stensby H Values Index

[0155] Lactic acid 90.08 3.20 14.62 55.82 76.25 Hydrochloric 90.21 3.16 11.52 65.13 77.01 acid

[0156] Citric acid 91.22 3.08 14.67 56.45 78.48 Acetic acid 91.49 3.11 14.44 57.50 79.31

[0157]

[0158] Example 4: Measurement of foaming capacity

[0159] Materials:

[0160] 5 g pea protein isolate from example 1

[0161] 300 ml distilled water

[0162] Thermomix with foam whisk attachment

[0163] 500 ml graduated cylinder

[0164] Spatula

[0165] Procedure:

[0166] 1. Measure 5 g of pea protein isolate and 300 ml of distilled water.

[0167] 2. Equip the Thermomix with the foam whisk attachment.

[0168] 3. Pour the distilled water into the Thermomix, followed by the pea protein isolate, ensuring minimal contact of the protein with the equipment.

[0169] 4. Set the Thermomix to speed 4 and run for 2 minutes.

[0170] 5. After 2 minutes, open the Thermomix and transfer the contents into a 500 ml graduated cylinder, using a spatula to scrape out any remaining mixture.

[0171] 6. Record the initial fill height (time 0), noting both the liquid level and the foam level.

[0172] 7. Measure the liquid fill height again at 5 minutes and 15 minutes.

[0173] The result are shown in Figure 2. It was surprisingly found that for protein isolate that used lactic acid, citric acid or acetic acid in the manufacture a higher increase in fluid (260 - 285 ml) could be observed after 5 min. For protein isolates that used HCI an increase of fluid of ca. 210 ml could be observed. When lactic acid was used in the manufacture, the protein isolate showed the highest increase in fluid (285 ml). A higher fluid formation is connected to a reduced foaming capacity.

[0174] Example 5: Measurement of gelling properties

[0175] Material

[0176] The following appliances were used for the gelling tests:

[0177] Vorwerk THERMOMIX TM6 with steamer attachment

[0178] Ultra Turrax IKA T50250 ml measuring cylinder

[0179] Petri dishes (glass, diameter 6 cm)

[0180] Silicone spatula

[0181] Metal spatula

[0182] Protein powder (Protein powder can be used as an equivalent for all protein extracts, isolates and the like, in the present experiment a protein isolate with a protein content of 85 % was used)

[0183] Sample preparation

[0184] For the 25 % gelation test, 5 g protein powder was stirred into 15 g water in a 250 ml measuring cylinder using a silicone spatula. Stirring was carried out until a homogeneous mass was obtained. The samples were then left to soak for one hour at room temperature. During this time, the samples were thoroughly dispersed after 30 min with the Ultra Turrax on level 4,5 for 30 s and after 60 min they were mixed with a spatula.

[0185] Steam treatment

[0186] After the soaking phase, the samples were steamed using the Thermomix TM6. For this purpose, the samples were transferred to glass Petri dishes and placed in the steam cooking attachment of the appliance. Steaming was carried out for 35 min at 95 °C. The samples were then cooled on ice for 15 min and stored at 6 °C.

[0187] Sample measurement

[0188] For the sample analysis the cooled gels were taken out of the fridge and directly placed on the Rheometer measuring plate. This was done with a metal spatula. Therefore, it was necessary to be careful not to destroy the gel. The remaining gel should be directly placed in the fridge again. The rheometer settings were saved in the rheometer method “Gelprotokoll Ultraturrax”. The settings were as follows:

[0189] Onset: temperature check 8°C

[0190] Measuring position: distance 60 mm

[0191] Final temperature check: Stability test 15 s, 8°C

[0192] Shear deformation: oscillating ramp 0.01 % to 100 %; circulating frequency 10 / sEvaluation linear viscosity range: linear viscoelastic region (LVR) suggestion 3%; LVR limit 1 %

[0193] Sample analysis

[0194] To compare different sample lines, the complex shear modulus at 1% strain within the Linear Viscoelastic (LVE) region was used as a reference point. This strain level ensures that the material response remains within the linear regime, allowing for a reliable comparison of intrinsic viscoelastic properties. Notably, at this point, the brittleness of the gel becomes apparent, as deviations in the complex modulus indicate structural weaknesses or reduced elasticity in certain formulations. This observation provides valuable insight into the mechanical stability and robustness of the gel network.

[0195] The results are shown in Table 3.

[0196] Table 3: Gelling properties at 25% gelling concentration

[0197] Acid G*

[0198] Lactic acid 917 ± 66.00

[0199] Hydrochloric acid 1030

[0200] Citric acid 1585 ± 49.50

[0201] Acetic acid 606 ± 21.92

[0202]

[0203] Example 6: Solubility Tests

[0204] The solubility test measures the solubility in a quantitative way by determining the mg soluble protein perg protein isolate.

[0205] Materials

[0206] Chemicals:

[0207] 1 M NaOH

[0208] 6 M HCI

[0209] Distilled water

[0210] Albumin Bovine Serum Fraction V (Storage at 4°C)ROTIQuant (Storage at 4°C)

[0211] Equipment:

[0212] 50 ml Falcon tubes, 5mL and 1 ,5mL Eppendorf tubes

[0213] Centrifuge: Eppendorf 5910 Ri cooled with Falcon inserts

[0214] Magnetic stirrer

[0215] Lab scale: PCE Instruments PCE-BSH 10000

[0216] pH-Meter Mettler Toledo Easy pH

[0217] Photometer Mettler Toledo

[0218] Sample preparation

[0219] Dissolution

[0220] 1. Dissolve 0.5 g of protein isolate in 50 mL of distilled water. Document any deviations in the weighed amount and recalculate the required amount of water if the weighed amount deviates from 0.5 g.

[0221] 2. Dissolve the protein isolate for 5min using the multiple magnetic stirrer.

[0222] 3. Adjust pH to 7 with NaOH (or HCI), while constantly stirring on magnetic stirrer. Be gentle when adjusting, use small amounts of base or acidic.

[0223] 4. After adjusting the pH, stir for 5 hours at RT (20°C) at 600rpm.

[0224] Centrifugation

[0225] 1. Transfer the solution to a 50 ml Falcon tube (document the weight of the empty and full Falcon tube).

[0226] 2. Centrifuge at 4500 rpm for 30 minutes at 20°C.

[0227] 3. Transfer the supernatant to a fresh Falcon tube (document the weight of the empty and full Falcon tube). The pellet is no longer needed for the solubility protocol. The weight measurements can be documented and compared over several samples for protocol check.

[0228] 4. Continue with the supernatant for the Bradford Assay.Bradford Assay

[0229] 1. Prepare the blank:

[0230] Pipette 800 pL of distilled water into a clean cuvette for the blank.

[0231] 2. Prepare the samples:

[0232] The protein isolates should be diluted 1:250. Therefore, they are prediluted 1:10 in

[0233] Eppendorf tubes. Pipette 90pL of distilled water and add 10pL of sample. (or900pL + 100pL)

[0234] The samples are then diluted 1 :25, but this time directly in the cuvette. Therefore,

[0235] pipette 768pL of distilled water and add 32pL of sample to reach a total volume of 800 pL

[0236] for each sample. Before adding the sample make sure to vortex them properly.

[0237] 3. Prepare the BSA standard:

[0238] Concentration range: 2-25pg / mL (6-point calibration)

[0239] Prepare a stock solution 2mg / mL (weight in 5mg BSA dissolve in 2.5mL H2O).

[0240] Dilute the stock to 10Opg / mL by pipetting 50pL of the stock solution with 950pL of distilled water.

[0241] Dilute the calibration curve fresh every day according to the table (V 800pL) directly in the cuevettes. Use the 100pg / mL stock.

[0242] 4. Add ROTIQuant (dye reagent):

[0243] Mix the stock solution thoroughly before removing an aliquot and check the color -if it’s

[0244] not red anymore acidify it with orthophosphoric acid. Take it out of the fridge before starting the experiment, to equilibrate it to RT (20°C).

[0245] Aliquot the needed amount for the assay, to avoid pipetting out of the stock.Add 200 pL of ROTIQuant to each cuvette (including the blank), mix the solutions by pipetting up and down and immediately start the timer.

[0246] 5. Incubate & Mix:

[0247] Incubate the cuevettes for 10min in total.

[0248] After 5min mix the contents of each cuvette by inverting one time (do not shake)- wear gloves.

[0249] After 10min, directly before measuring, mix the contents of each cuvette by inverting several times (do not shake)- wear gloves.

[0250] 6. Measure:

[0251] Open the BRADFORD method and start with the blank sample by clicking on 0.0. Continue with the standards and samples by measuring the optical density at 595 nm (OD595) against the blank.

[0252] 7. Evaluate:

[0253] Plot the OD595 values of the standards against the amount of protein used (pg / mL). Create a standard curve (linear regression). Make sure to have proper linearity (R2>0.99).

[0254] Calculate the soluble protein of your samples using the standard curve and calculate the solubility according to the following equations:

[0255] fOD (sample) — i - - — - ixdilution factor

[0256] \m /

[0257] ■Soluble proteins Solublitity (94) = x 100

[0258] , Total protein }

[0259]

[0260] • OD(sample): Absorption measured with the photometer

[0261] b: y-axis intercept

[0262] m: slopedilution factor: 250 (or else)

[0263] Total protein (pg / mL): Estimated amount of total protein initially weighted in (~8500pg / mL)

[0264] The results are shown in Table 4.

[0265] Table 4: Solubility measurements

[0266] Acid mg soluble protein per g sample Lactic acid 781.53

[0267] Hydrochloric acid 607.52

[0268] Citric acid 608.83

[0269] Acetic acid 587.82

[0270]

Claims

Claims1. A method of producing a pea protein isolate having improved physicochemical properties of the pea protein ingredient of a Pisum sativum plant, the method comprising the following steps:i. providing at least one pea flour;ii. isolating the protein ingredient from the pea flour involving the steps of:a) mixing the provided pea flour from step i. with at least one buffer and adjusting the pH of the solution to a range from about 6.5 to 9.5;b) separating the soluble fraction and the solid fraction and obtaining a soluble and a solid fraction;c) providing at least one organic acid individually being selected from lactic acid, citric acid and acetic acid, or a combination thereof, to the soluble fraction, preferably wherein the organic acid at least includes citric acid;d) adjusting the pH to the isoelectric point of the pea protein ingredient, preferably in a range from about 4.0 to 5.0, and obtaining a soluble and a solid fraction, wherein the solid fraction contains the pea protein ingredient;Hi. optionally drying the obtained solid fraction and obtaining a pea protein isolate,wherein the pea protein ingredient is not heated to a temperature of 60 °C or higher, preferably 50 °C or higher, most preferably 40 °C or higher during step ii and / or before or during step Hi.

2. The method according to claim 1 , wherein the organic acid provided in step ii.c) is provided in a concentration of at least 40% vol. / vol., at least 45% vol. / vol., at least 50% vol. / vol., at least 55% vol. / vol., at least 60% vol. / vol., at least 65% vol. / vol., at least 70% vol. / vol., at least 75% vol. / vol., at least 80% vol. / vol., at least 85% vol. / vol., at least 90% vol. / vol., or at least 95% vol. / vol.,preferably at 60% vol. / vol. to 90% vol. / vol. and especially preferably 75% vol. / vol. to 85% vol. / vol..

3. The method according to claim 1 or 2, wherein the at least one buffer in step ii. a) is selected from water, ethanol, a solution of phosphates and its salts or mixtures thereof.

4. The method according to any one of the preceding claims, wherein the soluble and the solid fraction are separated in step ii. b) with a technique selected from the group consisting of sedimentation, centrifugation and / or filtration.

5. The method according to any of the preceding claims, wherein step ii. c) includes an incubation after addition of the at least one organic acid, preferably while stirring the mixture.

6. The method according to claim 5, wherein the incubation is performed for a duration of five to 90 minutes preferably ten to 60 minutes.

7. The method according to any one of claim 1 to 6, wherein step ii. d) includes a step of incubation of the mixture, preferably under stirring for a time of 1 to 90 minutes, preferably 1 to 60 minutes and especially preferably 1 to 30 minutes.

8. The method according to any one of claims 1 to 7, wherein the drying in step Hi. is done via spray-drying, freeze drying or thermal drying.

9. A pea protein isolate, preferably having at least one altered physicochemical property compared to a pea protein isolate of a Pisum sativum plant not obtained with a method according to any one of claims 1 to 8, obtained or obtainable by a method according to any one of claims 1 to 8.

10. The pea protein isolate according to claim 9, wherein the at least one altered physicochemical property is selected from the group consisting of taste, solubility, emulsion stability, color, gelling capacity, foaming capacity, thermal stability, protein composition and profile of the composition of volatile compounds.

11. The pea protein isolate according to claim 9 or 10, wherein the pea protein isolate has a higher solubility when lactic acid is provided in step ii c) or wherein the pea protein isolate has higher gel strength when citric acid is provided in step ii c), or wherein the pea protein has lower gelling capacity when acetic acid is provided in step ii c).

12. A mixture comprising a pea protein isolate of a Pisum sativum plant according to any one of claims 9 to 11 and at least one further additive and / or ingredient.

13. An alternative consumable product, or a hybrid composition or product thereof, comprising at least one pea protein isolate of a Pisum sativum plant according to any of claims 9 to 11 , or comprising at least one mixture of claim 12.

14. The alternative consumable product, or the hybrid composition or product thereof of claim 13, wherein the composition or product has a pH in the range of about 3.5 to about 8.5, preferably a pH from about 4.5 to about 8.

15. A use of a protein isolate of a Pisum sativum plant as defined in any of claims 9 to 11 , ora use of a mixture as defined in claim 12 for preparing an alternative consumable product or a hybrid composition or product thereof, preferably wherein the consumable product or hybrid composition or product is a food product or a cosmetic composition or product.

16. The use of claim 15, wherein a protein isolate of a Pisum sativum plant is obtained or obtainable by a method according to any one of claims 1 to 8, wherein lactic acid is provided in step ii c) and the pea protein isolate is used for preparing a drinkable alternative consumable product, in particular a shake or an ultra-heat treated drink, preferably a plant based milk analog, or wherein a protein isolate of a Pisum sativum plant is obtained or obtainable by a method according to any one of claims 1 to 8, wherein citric acid is provided in step ii c) and the pea protein isolate is used for preparing a gelled alternative consumable product, in particular a plant based yogurt or meat analog or dessert, or wherein a protein isolate of a Pisum sativum plant is obtained or obtainable by a method according to any one of claims 1 to 8, wherein acetic acid is provided in step ii c) and the pea protein isolate is used for preparing a pumpable or low viscosity alternative consumable product.

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