Plant protein isolate improved by means of whole cell biomass processing

Abstract

The present invention relates to a method for improving the organoleptic properties of a material rich in plant proteins, the method comprising the following steps: 1. suspending a material rich in plant proteins in a preferentially aqueous solvent so as to obtain a suspension having a dry matter content of between 5% and 20%; 2. adding one or more microbial strains to the suspension of step 1, wherein the one or more microbial strains are chosen from among lactic acid bacteria strains, yeast strains, or a mixture thereof, so as to obtain an inoculated suspension having a cell density of between 1.105 and 1.1010 cfu / mL; 3. incubating the suspension of step 2 at a temperature of between 20°C and 40°C for a duration of between 30 and 120 minutes. The invention also relates to a plant protein isolate that is obtainable by means of the method of the invention and to the use thereof.

Description

Description PLANT PROTEIN ISOLATE ENHANCED VIA WHOLE CELL BIOMASS PROCESSING Technical field

[0001] The invention relates to the field of vegetable proteins, in particular legume protein isolates, even more particularly pea protein isolates. Prior art

[0002] Human daily protein requirements are between 12 and 20% of the diet. These proteins are provided by both animal products (meat, fish, eggs, dairy products) and plant products (cereals, legumes, algae).

[0003] However, in industrialized countries, protein intake is predominantly in the form of animal protein. However, numerous studies show that excessive consumption of animal protein at the expense of plant protein is one of the causes of increased cancer and cardiovascular disease.

[0004] Furthermore, animal proteins have many disadvantages, both in terms of their allergenic potential, particularly concerning proteins from milk or eggs, and on an environmental level in relation to the harmful effects of intensive farming.

[0005] Thus, there is a growing demand from manufacturers for compounds of plant origin with interesting nutritional and functional properties without presenting the disadvantages of compounds of animal origin.

[0006] Soy was, and remains, the first plant-based alternative to animal protein. However, using soy does have some definite disadvantages. Soybeans are often of GMO origin, and obtaining their protein requires a solvent-based deoiling process.

[0007] Since the 1970s, grain legumes, including peas in particular, have grown significantly in Europe, mainly in France, as an alternative protein source to animal protein for animal and human food. Peas contain approximately 27% protein by weight. The term "pea" is considered here in its broadest sense and includes in particular all wild varieties of "smooth pea" and all mutant varieties of "smooth pea" and "wrinkled pea", regardless of the uses for which said varieties are generally intended (human food, animal nutrition and / or other uses). These seeds are non-GMO and do not require solvent deoiling.

[0008] Pea protein, mainly pea globulin, has been extracted and processed industrially for many years. Several processes exist that can be categorized into two main families: so-called "dry" processes and so-called "wet" processes. The former consist of the particle size reduction of legume seeds into flour followed by particle size separation using an ascending air current, a process commonly called turbo-separation. These processes lead to the production of protein concentrates whose protein content does not exceed 60%-70%. These processes are not to be considered within the scope of the present invention, which falls within the field of isolates whose protein concentration is higher than these levels.

[0009] Concerning the second family of so-called "wet" processes, we can cite, as an example of a process for extracting pea protein, patent EP 1 400 537. In this process, the seed is ground in the absence of water (a process known as "dry grinding") in order to obtain a flour. This flour will then be suspended in water in order to extract the protein by precipitation. Precipitation is carried out by adjusting the isoelectric pH and / or by heating the medium.

[0010] Such processes have several disadvantages, in particular the production of unwanted vegetable flavors.

[0011] Experts in the field have long developed several methods to improve this flavor.

[0012] First of all, we can cite the use of thermal processes during the extraction process and / or on the final product. Such a heat treatment will make it possible to eliminate the endogenous enzymes responsible for the appearance of these unwanted flavors (such as lipoxygenase) and / or the organovolatile compounds carrying these flavors (e.g. hexanal). These heat treatments, as well known for example in patent applications WO 2020 / 260841 or WO 2020 / 064822, nevertheless have several disadvantages, such as an additional cost of the process (due to the acquisition and maintenance of the appropriate equipment but also its use) or the denaturation of proteins potentially sensitive to heat treatment.

[0013] A second approach involves adding masking or flavoring compounds to the protein as soon as the protein is obtained during its manufacturing process or during its formulation mainly in a food. Here again, many problems persist, including in particular the additional cost due to the use of such compounds or the regulatory issues of using a chemical compound for these purposes.

[0014] An alternative approach consists of using bacterial strains to modulate the organoleptic profile. The strain(s) will be added during the extraction process (preferably at the end of the process, i.e. with the final product) and subjected to physicochemical conditions allowing their growth. During this growth, the strains, through the activation of their metabolisms, will consume several compounds including those responsible for the flavor and the organoleptic profile (e.g. hexanal). Here too, problems persist, such as the hydrolysis of proteins by proteases that are constitutive or produced by the strains, or the production of unwanted flavors due, for example, to the production of organic acids such as lactic acid.

[0015] Finally, we can cite patent application EP 0 255 588 which presents a use of acetic acid bacteria to reduce the hexanal content and therefore the associated plant flavor of food products, particularly in alcoholic beverages. Example 1 presents a use of the acetic acid bacterium Gluconobacter suboxydans on soy milk. The reduction of hexanal is observed and the sensory panel confirms the reduction of the "grass" flavor. It should be noted that the other flavors are unchanged and therefore other flavors such as bitterness or acid taste quite often noted in vegetable proteins remain unchanged and may even be reinforced due to the disappearance of hexanal.

[0016] It is to the applicant's credit to have worked in this field and to have discovered a set of strains making it possible to improve the organoleptic profile of a plant protein, as well as to have developed the process using it in order to obtain a protein isolate whose functional and organoleptic characteristics are optimized to a level never before achieved. In addition, the strains and the process according to the invention using them make it possible to obtain a plant protein isolate whose organoleptic performances are of definite interest for the agri-food industry.

[0017] The invention will be better understood in the descriptive part of the present application which follows. General description

[0018] The present invention relates to a method for improving the organoleptic properties of a material rich in vegetable proteins comprising the following steps 1. suspending a material rich in vegetable proteins in a preferably aqueous solvent so as to obtain a suspension having a dry matter content of between 5% and 20%, preferably between 7.5% and 15%, even more preferably between 8% and 12%; 2. the addition of one or more microbial strains to the suspension from step 1, the microbial strain(s) being chosen from strains of lactic acid bacteria, strains of yeast, or a mixture thereof, the strain(s) preferably being one or more strains of lactic acid bacteria, so as to obtain a seeded suspension having a cell density of between 1.10 5 and 1.10 10 cfu / mL, preferably between 1.10 7 and 1.10 9 cfu / mL, preferably between 5.10 7 and 5.10 8 cfu / mL; 3. incubation of the suspension from step 2 at a temperature between 20°C and 40°C for a time between 30 and 120 minutes, preferably between 45 and 75 minutes, even more preferably between 50 and 70 minutes.

[0019] In a preferred embodiment, the method according to the invention is characterized in that the material rich in vegetable proteins from step 1 is in the form of a powder containing a quantity of vegetable protein greater than 1% by weight, preferably between 70% and 90% by weight, relative to the weight of powder. In this embodiment, the powder is preferably essentially derived from seeds, typically legume seeds such as pea or field bean seeds, having been ground and reduced to a powder state. In other words, the powder is preferably a flour. The seeds may of course have undergone preparation steps including, but not limited to, cleaning, sorting, removal of the outer shell (also called "dehulling"), and heat treatment.

[0020] In a preferred embodiment, the method according to the invention is characterized in that the material rich in vegetable protein from step 1 is an isolate or a concentrate, preferably an isolate whose protein content is greater than 70% by dry weight on isolate dry matter, preferably between 75% and 95% by dry weight on isolate dry matter. The protein content values ​​are potentially 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 or 95%. As well as any range included between two of each of these values. Advantageously, the material rich in vegetable proteins from step 1 is a vegetable protein isolate or concentrate, preferably a vegetable protein isolate whose protein content is greater than 80% by dry weight, preferably between 80% and 90% by dry weight, relative to the dry weight of isolate.

[0021] In a preferred embodiment, the method according to the invention is characterized in that the material rich in vegetable protein from step 1 comes from a legume, preferably from a legume selected from the group consisting of peas and field beans, more preferably from peas.

[0022] In a preferred embodiment, the method according to the invention is characterized in that the pH of the suspension of material rich in vegetable protein from step 1 is rectified so as to be between 5.0 and 7.5, preferably between 6.5 and 7.5; preferably so as to be equal to 7.0, before implementing step 2.

[0023] In a preferred embodiment, the method according to the invention is characterized in that the sugar content of the suspension of material rich in vegetable proteins from step 1 is rectified so as to contain between 1% and 10% of sugars, preferably between 2% and 8%, even more preferably between 3% and 6%.

[0024] In a preferred embodiment; the method according to the invention is characterized in that the strain(s) of step 2 are selected from a strain of Limosilactobacillus fermentum, Limosilactobacillus reuteri, Lacticaseibacillus rhamnosus or Saccharomyces cerevisiae, preferentially Limosilactobacillus fermentum, Limosilactobacillus reuteri, or Lactobacillus rhamnosus. As explained below, the terms Limosilactobacillus and lacticaseibacillus can be interchanged with the term Lactobacillus due to a change in taxonomy (see the article Zheng et al., “A taxonomy note on the genus Lactobacillus: Description of 23 novel genera, emended description of the genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae” (, Int. J. Syst. Evol. Microbiol. 2020; 70:2782-2858 DOI 10.1099 / ijsem.0.004107).The strain(s) for step 2 can therefore be preferentially selected from Limosilactobacillus fermentum, Limosilactobacillus reuteri, or Lacticaseibacillus rhamnosus.

[0025] In a preferred embodiment, the strain(s) used during step 2 of the method of the invention have been cultured according to a method comprising: i. inoculation of a solid culture medium, for example agar, with a sample of the strain(s); ii. pre-cultivation of the strain(s), by incubation of the inoculated solid culture medium from step 1 in a non-shaken incubator, preferably for between 10h and 24h; iii. propagation of the strain, by inoculation of a liquid culture medium with a sample of pre-cultivated strain from step ii, and culturing the strain in liquid medium.

[0026] In one embodiment, the incubation time of step 11 is between 10 h and 20 h.

[0027] In a preferred embodiment, the method according to the invention is characterized in that step 3 is followed by a step 4 comprising the following sub-steps 4a. optionally, rectification of the pH of the suspension obtained at the end of step 3 to a pH between 5 and 9, preferably between 6 and 9, preferably a pH of 7, preferably by adding soda or lime to said suspension; 4b. heat treatment of the suspension from step 3 or step 4a between 100°C and 160°C for 0.1 to 1 s, and 4c. drying the suspension from step 4b using an atomizer until a vegetable protein isolate is obtained, preferably having a dry matter content greater than 95% relative to the mass of isolate.

[0028] The invention also relates to a plant protein isolate obtainable by the method of the invention.

[0029] In one embodiment, the plant protein isolate capable of being obtained by the process of the invention is characterized in that its degree of hydrolysis (or DH) is less than 10%, preferably less than 5%.

[0030] In one embodiment, the plant protein isolate obtainable by the method of the invention is characterized in that its protein content is between 70% and 95% by weight, preferably between 82% and 92% by weight, preferably between 84% and 90% by weight, preferably between 84% and 88% by weight, relative to the total dry matter weight of the isolate. The protein content values ​​are potentially 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 or 95%. As well as any range included between two of each of these values.Preferably, the plant protein isolate obtainable by the process of the invention is characterized in that its protein content is between 80% and 95% by weight, preferably between 82% and 92% by weight, preferably between 84% and 90% by weight, preferably between 84% and 88% by weight, relative to the total dry matter weight of the isolate.

[0031] The invention also relates to the use of an isolate obtainable by the process of the invention or obtained according to the process of the invention for industrial applications including food, nutraceutical and pharmaceutical applications.

[0032] The invention will be better understood by reading the detailed description below. Detailed description

[0033] The present invention relates to a method for improving the organoleptic properties of a material rich in vegetable proteins comprising the following steps 1. suspending a material rich in vegetable proteins in a preferably aqueous solvent so as to obtain a suspension having a dry matter content of between 5% and 20%, preferably between 7.5% and 15%, even more preferably between 8% and 12%; 2. the addition of one or more microbial strains to the suspension from step 1, the microbial strain(s) being chosen from strains of lactic acid bacteria, strains of yeast, or a mixture thereof, the strain(s) preferably being one or more strains of lactic acid bacteria, so as to obtain a seeded suspension having a cell density of between 1.10 5 and 1.10 10 cfu / mL, preferably between 1.10 7 and 1.10 9cfu / mL, preferably between 5.10 7 and 5.10 8 cfu / mL; 3. incubation of the suspension from step 2 at a temperature between 20°C and 40°C for a time between 30 and 120 minutes, preferably between 45 and 75 minutes, even more preferably between 50 and 70 minutes.

[0034] By "organoleptic" we mean all the stimuli that relate to the sensory sphere including taste, flavor, and smell. The organoleptic profile of a compound and / or food can be evaluated using a sensory panel. A sensory panel is made up of a group of people who will taste the compound and / or food according to a well-defined protocol, then each member of the panel will give their sensory evaluation, typically composed of a descriptor (e.g. bitter, sweet, pea taste, vegetable flavor, cardboard) and a score indicating the intensity of perception of this descriptor.

[0035] By "improving organoleptic properties" or "improving organoleptic qualities" is meant in the present invention a reduction undesirable stimuli associated with plant protein-rich materials (for example, associated with plant protein isolates), such as plant flavors, typically herbaceous flavors or pea flavors ("beany" flavors). In the context of the present invention, this reduction of undesirable stimuli is enabled by the conversion of compounds such as aldehydes, ketones and thiols present in the plant protein-rich material into other compounds (alcohols and / or carboxylic acids) under the action of one or more microbial strains as described in the present application. Indeed, these aldehydes, ketones and thiols are the main contributors of these undesirable stimuli, and the products of their bioconversions have, in particular, flavors or odors less characteristic of plant protein-rich materials, and / or higher thresholds of perception by the consumer.For example, reducing the quantity of aldehydes with green notes (“green-note aldehydes”) leads to an organoleptic improvement of the products, through a reduction in “plant” sensory attributes.

[0036] In one embodiment, the method for improving the organoleptic properties of plant proteins of the invention is a method for converting aldehydes, ketones, and / or thiols present in plant protein-rich materials into other compounds having flavors or odors less characteristic of plant proteins and / or higher perception thresholds.

[0037] In one embodiment, the method for improving the organoleptic properties of plant proteins of the invention is a method for converting all or part of the aldehydes having green notes present in the plant proteins into other compounds.

[0038] Plant protein means any composition extracted from a natural raw material such as seeds and preferably modified to make it more available and effective in its formulation for the ultimate purpose of consumption. Modifications can be very limited (e.g., simple grinding of the seed) or very complex (e.g., wet-process manufacturing of an isolate).

[0039] By "material rich in vegetable proteins" is meant any powder, solution, floc containing at least 1%, preferably 25%, by weight of proteins, relative to the total weight of material rich in vegetable proteins. Mention may be made, without limitation, of flours, concentrates, isolates, seeds.

[0040] Lactic acid bacteria are all Gram-positive, anaerobic, partially oxygen-tolerant bacteria that generally do not produce spores, are cocci or rod-shaped, and are capable of fermenting sugars into lactic acid. According to current taxonomic classification, they belong to the phylum Firmicutes, the class Bacilli, and the order Lactobacillales, which includes the following families: Aerococcaceae, Carnobacteriaceae, Enterococcaceae, Lactobacillaceae, Leuconostocaceae, and Streptococcaceae. Bifidobacteriaceae of the order Bifidobacteriales are also classified as lactic acid bacteria. Within the family Lactobacillacea, the genus Lactobacillus is particularly important. The terms Limosilactobacillus and lacticaseibacillus may be interchanged with the term Lactobacillus due to a change in taxonomy (see the article Zheng et al., 'A taxonomy note on the genus Lactobacillus'.Description of 23 novel genera, amended description of the genus Lactobacillus Beijerinck 1901 , and union of Lactobacillaceae and Leuconostocaceae », Int. J. Syst. Evol. Microbiol. 2020;70:2782-2858 DOI 10.1099 / ijsem.0.004107). Thus Lactobacillus fermentum is now equivalent to Limosilactobacillus fermentum; Lactobacillus rhamnosus is now equivalent to Lacticaseibacillus rhamnosus; Lactobacillus reuteri is now equivalent to Limosilactobacillus reuteri).

[0041] By "yeasts" we mean all unicellular fungi of variable shape depending on the species (spherical, ovoid or elliptical, bottle-shaped, triangular or apiculate (swollen at each end like a lemon) but generally oval, of about 6 to 10 microns and up to 50 microns, multiplying by budding or by fission (fissiparity). Most are related to the Ascomycetes (truffle type, pézize), some to the other large group of higher fungi, the Basidiomycetes (amanita type, boletes) and others finally are imperfect forms not clearly assignable to a defined group. They are often capable of sporulation either for the purpose of dormancy in an unfavorable environment, or for the purpose of dispersal. The common term yeast generally refers to the genus Saccharomyces (brewer's yeast or baker's yeast). There are many other genera of yeast; among the best known are the genus Candida and Pichia.

[0042] The term "cfu" refers to "colony forming unit" and is used to estimate the number of viable bacteria or fungal cells in a sample. Viability is defined as the ability to multiply by cell division under controlled conditions.

[0043] Cell density refers to the amount of bacteria or fungal cells present in a sample. It can be expressed in cfu / mL

[0044] Suspension step 1 consists of mixing a material rich in vegetable protein in a preferably aqueous solvent, so as to obtain a suspension of vegetable protein reaching between 5% and 20% of dry matter, preferably between 7.5% and 15%, even more preferably between 8% and 12%. The values ​​will therefore potentially be 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24% or 25%. As well as any range included between two of each of these values.

[0045] In the present invention, the expression "protein content" or "protein richness", in particular relating to a protein-rich material or a composition according to the invention, designates the quantity of nitrogen, typically measured by the Kjeldhal method, multiplied by 6.25 and expressed as % by total weight of protein-rich material or composition according to the invention.

[0046] In a preferred embodiment, the method according to the invention is characterized in that the material rich in vegetable protein from step 1 is in the form of a vegetable powder commonly called flour containing a protein content greater than 1% by weight, preferably between 10% and 40% by weight, relative to the weight of powder, preferably between 20% and 30%.

[0047] In this method, the powder is preferably essentially made up of seeds that have been ground and reduced to a powder state. The seeds may have obviously undergone preparation steps including, but not limited to, cleaning, sorting, removal of the outer shell (also called "dehulling"), and heat treatment.

[0048] By "isolate" is meant a protein-rich material whose protein content is greater than 70% by dry weight, preferably greater than 80% by dry weight, even more preferably between 80% and 90% by dry weight, relative to the total dry matter of the isolate.

[0049] By "concentrate" is meant a protein-rich material with a protein content of between 25% and 70% by dry weight, preferably between 35% and 60% by dry weight, even more preferably between 40% and 50% by dry weight, relative to the total dry matter of the concentrate.

[0050] In a preferred embodiment, the method according to the invention is characterized in that the material rich in vegetable protein from step 1 is an isolate or a concentrate, preferably an isolate whose protein content is greater than 70% by dry weight on isolate dry matter, preferably between 75% and 95% by dry weight on isolate dry matter. The protein content values ​​are potentially 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 or 95%. As well as any range included between two of each of these values.

[0051] In a preferred embodiment, the method according to the invention is characterized in that the material rich in vegetable protein from step 1 comes from a legume, preferably comes from a legume selected from the group consisting of peas and field beans, more preferably comes from peas.

[0052] Legumes are the dicotyledonous family of plants in the order Fabales. It is one of the largest families of flowering plants, third only to Orchidaceae and Asteraceae in terms of the number of species. It has approximately 765 genera comprising more than 19,500 species. Several legumes are important cultivated plants, including soybeans, beans, peas, faba beans, chickpeas, peanuts, lentils, alfalfa, various clovers, broad beans, carob, and licorice.

[0053] By "pea" is meant its broadest definition and including in particular all varieties of "smooth pea" and "wrinkled pea", and all mutant varieties of "smooth pea" and "wrinkled pea", and this, whatever the uses for which said varieties are generally intended (human food, animal nutrition and / or other uses).

[0054] The term "pea" in the present application includes pea varieties belonging to the genus Pisum and more particularly to the species sativum and aestivum. Said mutant varieties are in particular those called "r mutants", "rb mutants", "rug 3 mutants", "rug 4 mutants", "rug 5 mutants" and "lam mutants" as described in the article by CL HEYDLEY et al. entitled "Developing novel pea starches" Proceedings of the Symposium of the Industrial Biochemistry and Biotechnology Group of the Biochemical Society, 1996, pp. 77-87.

[0055] The term "fava bean" refers to the group of annual plants of the species Vicia faba, belonging to the group of legumes of the family Fabaceae, subfamily Faboideae, tribe Fabeae. A distinction is made between Minor and Major varieties. In the present invention, wild varieties and those obtained by genetic engineering or varietal selection are all excellent sources.

[0056] By "preferably aqueous solvent" is meant any solvent whose composition is predominantly water. By predominantly is meant that more than 50% by weight, preferably more than 90% by weight, even more preferably more than 95%, of the aqueous solvent is water, by weight of solvent, or even the aqueous solvent is "pure" water, that is to say a solvent composed of 100% by weight of water.

[0057] By "water" is meant any so-called potable water that is suitable for this purpose. By "potable water" is meant water that can be drunk or used for domestic and industrial purposes without risk to health. Preferably, its conductivity is chosen between 400 and 1100, preferably between 400 and 600 pS / cm. More preferably in the present invention, it will be understood that this potable water has a sulfate content of less than 250 mg / l, a chloride content of less than 200 mg / l, a potassium content of less than 12 mg / l, a pH between 6.5 and 9 and a TH (Hydrometric Title, i.e. the hardness of the water, which corresponds to the measurement of the content of calcium and magnesium ions in water) greater than 15 French degrees. In other words, potable water must not have less than 60 mg / l of calcium or 36 mg / l of magnesium. This definition includes drinking water, decarbonated water, and demineralized water.

[0058] The suspension is therefore obtained by intimately mixing the material rich in vegetable proteins and the aqueous solvent. There are several ways of proceeding including the introduction of a powder of material rich in vegetable proteins into the aqueous solvent (dry grinding followed by suspension) or the introduction of the material rich in vegetable proteins into the aqueous solvent followed by a grinding step (suspension followed by wet grinding).

[0059] This mixture is made so as to obtain a suspension of vegetable protein having a dry matter content (DM value) of between 5% and 20%, preferably between 7.5% and 15%, even more preferably between 8% and 12%. The dry matter content is measured by any method well known to those skilled in the art. It describes the percentage of solid in a mixture of substances. The higher the dry matter content, the drier the mixture.

[0060] Preferably, the so-called "desiccation" method is used. It consists of determining the quantity of water evaporated by heating a known quantity of a sample of known mass. A previously weighed sample (called fresh mass) is heated. Heating is continuous until the mass stabilizes, indicating that the evaporation of the water is complete. Preferably, the temperature used is 105°C to allow the residual water to evaporate. The dry residue is weighed and expressed as a ratio. The dry matter content of the analyzed sample is obtained.

[0061] Step 2 consists of adding one or more microbial strains to the suspension of raw material rich in vegetable protein from step 1, the strain(s) being selected from the group consisting of lactic acid bacteria or yeasts, preferably lactic acid bacteria, and the cell density of the suspension after introduction of the strain(s) being between 1.10 5 and 1.10 10 cfu / mL, preferably between 1.10 7 and 1.10 9 cfu / mL, preferably between 5.10 7 and 5.10 8 cfu / mL; The cell density could therefore be 1.10 5 cfu / mL, 2.10 5 cfu / mL, 3.10 5 cfu / mL, 4.10 5 cfu / mL, 5.10 5 cfu / mL, 6.10 5 cfu / mL, 7.10 5 cfu / mL, 8.10 5 cfu / mL, 9.10 5 cfu / mL, 1.10 6 cfu / mL, 2.10 6 cfu / mL, 3.10 6 cfu / mL, 4.10 6 cfu / mL, 5.106 cfu / mL, 6.10 6 cfu / mL, 7.10 6 cfu / mL, 8.10 6 cfu / mL, 9.10 6 cfu / mL, 1.10 7 cfu / mL, 2.10 7 cfu / mL, 3.10 7 cfu / mL, 4.10 7 cfu / mL, 5.10 7 cfu / mL, 6.10 7 cfu / mL, 7.10 7 cfu / mL, 8.10 7 cfu / mL, 9.10 7 cfu / mL, 1.10 8 cfu / mL, 2.10 8 cfu / mL, 3.10 8 cfu / mL, 4.10 8 cfu / mL, 5.10 8 cfu / mL, 6.10 8 cfu / mL, 7.10 8 cfu / mL, 8.10 8 cfu / mL, 9.10 8 cfu / mL, 1.10 9 cfu / mL, 2.10 9 cfu / mL, 3.10 9 cfu / mL, 4.10 9 cfu / mL, 5.10 9 cfu / mL, 6.10 9 cfu / mL, 7.10 9 cfu / mL, 8.10 9 cfu / mL, 9.10 9 cfu / mL, 1.10 10 cfu / mL, 2.10 10 cfu / mL, 3.10 10 cfu / mL, 4.10 10 cfu / mL, 5.10 10 cfu / mL, 6.10 10cfu / mL, 7.10 10 cfu / mL, 8.10 10 cfu / mL, 9.10 10 cfu / mL. As well as any range included between two of each of these values.

[0062] In a first mode, a single microbial strain is introduced into the suspension of raw material rich in vegetable protein from step 1.

[0063] In a second mode, two or more microbial strains are introduced at the same time into the suspension of raw material rich in vegetable protein from step 1.

[0064] In a third mode, two or more microbial strains are introduced consecutively into the suspension of raw material rich in vegetable protein from step 1.

[0065] To do this, the lactic acid bacteria or yeasts are cultivated in a first sub-step called culture in order to achieve a higher cell density than that described in the present application. The person skilled in the art will know how to adapt the culture conditions to the selected strain in order to achieve this density. The culture step is itself subdivided into two steps: a first pre-culture step aimed at reviving the stored strain (e.g. frozen or freeze-dried) and a second propagation step aimed at multiplying the strain to achieve the desired cell density.

[0066] According to one embodiment, the strain(s) used during step 2 have been cultured according to a method comprising: i. inoculation of a solid culture medium, for example agar, with a sample of strain, preferably frozen or freeze-dried strain; ii. pre-cultivation of the strain, by incubation of the inoculated solid culture medium from step 1 in a non-shaken incubator, preferably for 15 to 24 h; iii. propagation of the strain, by inoculation of a liquid culture medium with a sample of pre-cultivated strain from step ii, and culture of the strain in liquid medium.

[0067] Pre-cultivation and propagation are preferably carried out at a temperature appropriate to the strain: typically at a temperature of about 35°C-38°C, preferably 38°C for lactic acid bacteria and typically about 28-32°C for yeasts. Advantageously, the culture time of the propagation step 11 is relatively long, typically between 12 and 20 hours, preferably about 15 hours; and the propagation time is between 8 hours and 15 hours, and is preferably at least about 10 hours. Indeed, as shown in the experimental part, a minimum propagation time of 10 hours is preferable to obtain the maximum technical effect of the invention.

[0068] A preferred culture protocol is as follows: i. Seeding step: A frozen strain sample is taken into a cryotube and inoculated into a suitable culture medium, typically agar, with the culture medium being contained in a tube, for example in a 10 mL tube; ii. Pre-culture step: The tube is incubated in a non-shaken incubator for 15 to 24 h. iii. Propagation step: The pre-culture tube is then used to inoculate at 1% v / v a container (flask or Erlenmeyer flask), containing e.g. 1 L of medium for the above example of a 10 mL tube. Cultivation takes place at the optimal temperature for each strain for the optimal time for each, such as those detailed for lactic acid bacteria and yeast examples in Table 1.

[0069] The second sub-step then consists of introducing a determined quantity of the culture into another quantity of the suspension rich in vegetable proteins prepared in step 1 in order to finally obtain a cell density of the suspension between 1.10 5 and 1.10 10 cfu / mL, preferably between 1.10 7 and 1.10 9 cfu / mL, preferably between 5.10 7 and 5.10 8 cfu / mL.

[0070] For example, 10 mL of a lactic acid bacteria culture titrating 1.10 9 cfu / mL introduced into 90 mL of suspension rich in plant proteins will allow obtaining a 100 mL suspension having a cell density of 1.10 8 cfu / mL

[0071] In a preferred embodiment; the method according to the invention is characterized in that the strain(s) of step 2 are selected from a strain of Limosilactobacillus fermentum, Limosilactobacillus reuteri, Lacticaseibacillus rhamnosus or Saccharomyces cerevisiae, preferentially of Limosilactobacillus fermentum, Limosilactobacillus reuteri, or Lactobacillus rhamnosus. The terms Limosilactobacillus and lacticaseibacillus also mean Lactobacillus due to a change in taxonomy (see the article Zheng et al., Int. J. Syst. Evol. Microbiol. 2020;70:2782-2858 DOI 10.1099 / ijsem.0.004107). In a preferred embodiment, several strains such as those described above can be introduced together into the suspension. The strain(s) of step 2 can therefore be preferentially selected from Limosilactobacillus fermentum, Limosilactobacillus reuteri, or Lacticaseibacillus rhamnosus.

[0072] In a first mode, a single microbial strain selected from Limosilactobacillus fermentum, Limosilactobacillus reuteri, Lacticaseibacillus rhamnosus or Saccharomyces cerevisiae, preferentially from Limosilactobacillus fermentum, Limosilactobacillus reuteri, or Lactobacillus rhamnosus is introduced into the suspension of raw material rich in vegetable protein from step 1.

[0073] In a second mode, two or more microbial strains selected from Limosilactobacillus fermentum, Limosilactobacillus reuteri, Lacticaseibacillus rhamnosus or Saccharomyces cerevisiae, preferably from Limosilactobacillus fermentum, Limosilactobacillus reuteri, or Lactobacillus rhamnosus are introduced at the same time into the suspension of raw material rich in vegetable protein from step 1.

[0074] The combination of strains can thus be - Limosilactobacillus fermentum with Limosilactobacillus reuteri, - Limosilactobacillus fermentum with Lacticaseibacillus rhamnosus, - Limosilactobacillus fermentum with Saccharomyces cerevisiae, - Limosilactobacillus reuteri with Lacticaseibacillus rhamnosus, - Limosilactobacillus reuteri with Saccharomyces cerevisiae, - Lacticoseibacillus rhamnosus with Saccharomyces cerevisiae, Limosilactobacillus fermentum with Limosilactobacillus reuteri and Lacticoseibacillus rhamnosus - Limosilactobacillus fermentum, Limosilactobacillus reuteri, and Saccharomyces cerevisiae - Limosilactobacillus fermentum, Lacticaseibacillus rhamnosus and Saccharomyces cerevisiae - Limosilactobacillus reuteri, Lacticaseibacillus rhamnosus and Saccharomyces cerevisiae - Limosilactobacillus fermentum, Limosilactobacillus reuteri, Lacticaseibacillus rhamnosus and Saccharomyces cerevisiae

[0075] In a third mode, two or more microbial strains selected from Limosilactobacillus fermentum, Limosilactobacillus reuteri, Lacticaseibacillus rhamnosus or Saccharomyces cerevisiae, preferably from Limosilactobacillus fermentum, Limosilactobacillus reuteri, or Lactobacillus rhamnosus are introduced consecutively into the suspension of raw material rich in vegetable protein from step 1.

[0076] The combination of strains can thus be - Limosilactobacillus fermentum then Limosilactobacillus reuteri, as well as the reverse - Limosilactobacillus fermentum then Lacticaseibacillus rhamnosus, as well as the reverse - Limosilactobacillus fermentum then Saccharomyces cerevisiae, as well as the reverse - Limosilactobacillus reuteri then Lacticaseibacillus rhamnosus, as well as the reverse - Limosilactobacillus reuteri then Saccharomyces cerevisiae, as well as the reverse - Lacticaseibacillus rhamnosus then Saccharomyces cerevisiae, as well as the reverse

[0077] Examples of such strains from reference collections are Lactobacillus fermentum CIP 102980 T or CNCM I-5802 (now Limosilactobacillus fermentum); Lactobacillus rhamnosus LMG 18243 or ATCC 53103, also known as Lacticaseibacillus rhamnosus GG (now Lacticaseibacillus rhamnosus); Lactobacillus reuteri DSM 17509 (now Limosilactobacillus reuteri); Sacharomyces cerevisiae CBS 8066.

[0078] Preferred culture parameters are described in Table 1: [Table 1]

[0079] (MRS = De Man Rogosa and Sharpe agar; MW = Malt Wickerham agar)

[0080] As will be exemplified further below in the examples section of the description, propagation of less than 10 hours or more than 20 hours will not allow the maximum expected effects to be obtained. Surprisingly, the holder discovered that this cultivation time was necessary for the strain(s) to be active.

[0081] Preferably at the end of the first culture period and before the second step of introducing the quantity thereof into the plant protein suspension, a wash is applied to the culture in order to remove the excess culture medium. The culture is centrifuged at 4000 G, at 4 °C for 10 min. The supernatant is removed and the pellet is resuspended in water. A new centrifugation is then carried out and the supernatant removed. The strains are then resuspended in a volume of water, depending on the calculated biomass, in order to be able to inoculate the test medium between 10 6 and 10 9, preferably between 10 7 and 10 8 , even more preferably at 10 8 CFU / mL (i.e. 10 9 CFU / mL if a suspension is inoculated at 10% aiming for a final cell density of 10 8 CFU / mL).

[0082] Contact step 3 consists of incubating the seeded suspension from step 2 at 30°C + / - 10°C for a time between 30 and 120 minutes, preferably between 45 and 75 minutes, even more preferably between 50 and 70 minutes. The temperatures may be 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C. As well as any range included between two of each of these values. Contact times 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C or 70°C. As well as any range included between two of each of these values.

[0083] Surprisingly and unexpectedly, the applicant discovered that a contact time of between 30 min and 120 min was sufficient to reduce undesirable flavors but also to bring out new desired flavors and, moreover, to limit the alteration of the protein such as, for example, enzymatic hydrolysis.

[0084] The person skilled in the art would never have taken into consideration a short time to carry out these transformations because this time corresponds to the latency period necessary for a strain to adapt before resuming catabolism. The very low quantities of sugars also seem too low for an efficient resumption of catabolism. The volatile compounds being extracellular must come into contact with the intracellular enzymes, be modified and then be released. This transport mechanism is not currently clearly identified but its complexity seems incompatible with the short biotransformation duration according to the invention.

[0085] Incubation can be static or agitated, in the latter case the agitation must be sufficient to keep the suspension homogeneous but not too high as this could degrade the cells.

[0086] In a preferred embodiment, the method according to the invention is characterized in that the pH of the suspension of material rich in vegetable protein from step 1 is rectified so as to be between 5.0 and 7.5, preferably between 6.5 and 7.5; preferably so as to be equal to 7.0, before implementing step 2. The pH may therefore be 5.0; 5.1; 5.2; 5.3; 5.4; 5.5; 5.6; 5.7; 5.8; 5.9; 6.0; 6.1; 6.2; 6.3; 6.4; 6.5; 6.6; 6.7; 6.8; 6.9; 7.0; 7.1; 7.2; 7.3; 7.4 or 7.5. As well as any range included between two of each of these values.

[0087] In a preferred embodiment, the method according to the invention is characterized in that the suspension of material rich in vegetable protein from step 1 contains between 0.1 and 1% of total sugars, preferably between 0.2% and 0.8%, even more preferably between 0.3% and 0.6%. The sugar values totals may be 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1.0%. As well as any range included between two of each of these values.

[0088] By "sugars" is meant a set of carbohydrates, for example selected from monosaccharides such as glucose, fructose, ribose, galactose, and mixtures thereof and oligosaccharides such as raffinose, sucrose, melezitose, stachyose and mixtures thereof.

[0089] For the purposes of the present invention, the term "monosaccharides" means carbohydrates or carbohydrates composed of a single molecule such as glucose, fructose, mannose, galactose, xylose, arabinose.

[0090] For the purposes of the present invention, the term "oligosaccharides" means oligomers formed from a number n of oses (monosaccharides) by alpha or beta glycosidic bond. By convention, the number n varies from 3 to 10. There, they are placed between the simple oses (n=1) and the polyosides (polysaccharides) (n>10). However, this limit of 10 units is not completely fixed and polyosides with a degree of polymerization of 11 to 25 are often assimilated to them. Oligosides comprising 2 oses are diholosides (sucrose), 3 oses are triholosides (raffinose, melezitose) and 4 oses are tetraholosides (stachyose). Oligosides can be linear (stachyose), branched or cyclic (cyclodextrin).

[0091] By "total sugars" is meant by the present invention the monosaccharide composition obtained after total hydrolysis of the sugars. The oligosaccharides and polysaccharides are completely hydrolyzed chemically (acid or base) and / or enzymatically (amylase), then the hydrolyzed sample is analyzed to determine the composition of the monosaccharides obtained.

[0092] In a preferred embodiment, the total sugars comprise a mixture of glucose, fructose, mannose, galactose, xylose and arabinose. Preferably, the total sugars comprise between 20% and 30% glucose, between 20% and 30% galactose and between 20% and 30% arabinose. For example, a typical composition of total sugars suitable for the invention is a mixture of 0.15 g / L glucose, 0.058 g / L fructose, 0.03 g / L mannose, 0.12 g / L galactose, 0.03 g / L xylose, 0.19 g / L arabinose and 0.003 g / L raffinose.

[0093] The total sugars present in the plant protein-rich material suspension from step 1 come partly from a mixture of sucrose, raffinose, melezitose and stachyose. The preferred content of raffinose and stachyose is between 0.02% and 0.05%, preferably between 0.03% and 0.04%.

[0094] It should be noted that not all isolates contain these sugars, in particular the mixture of sucrose, raffinose, melezitose and stachyose. Application EP 3 071 046 B1, for example, presents an extraction process including a grain steeping step allowing the almost total elimination of sugars including GOS (galactooligosaccharides) from the pea.

[0095] A step 4 comprising sub-steps can then also be carried out. These sub-steps aim to isolate the plant protein, extract it and stabilize it for its subsequent use.

[0096] Thus, in one embodiment, step 3 of the method according to the invention is followed by a step 4 comprising the following sub-steps: 4a. optionally, rectification of the pH of the suspension obtained at the end of step 3 to a pH between 5 and 9, preferably between 6 and 9, preferably a pH of 7, preferably by adding soda or lime to said suspension; 4b. heat treatment of the suspension from step 3 or step 4a between 100°C and 160°C for 0.1 to 1 s, and 4c. drying the suspension from step 4b using an atomizer until a vegetable protein isolate is obtained, preferably having a dry matter content greater than 95% relative to the mass of isolate.

[0097] Step 4b typically corresponds to a HTST or UHT type heat treatment.

[0098] Starting from a powder or flour, step 4 can for example include the following sub-steps: 4a. removal of insoluble fractions (mainly containing starch and fibers by internal centrifugation), 4b. isoelectric precipitation of the globulin fraction and its recovery by centrifugation, 4c. neutralization of pH to 7, 4d. HTST or UHT type heat treatment; 4th. drying using an atomizer.

[0099] In a preferred embodiment starting from an isolate, the method according to the invention is characterized in that step 3 is followed by the following steps: 4a. the optional rectification of the pH of the suspension obtained at the end of step 3 to a pH between 6 and 9, preferably a pH of 7, preferably by adding soda or lime to said suspension; 4b. the optional heat treatment of the suspension of step a between 100°C and 160°C for 0.1 to 1s, and 4c. drying the vegetable flour suspension from step b until a vegetable flour isolate with a dry matter content greater than 95% is obtained using an atomizer.

[0100] The invention also relates to a plant protein isolate obtainable by the method of the invention.

[0101] The invention also relates to a plant protein isolate, capable of being obtained by the process of the invention, characterized in that its degree of hydrolysis (or DH) is less than 10%, preferably less than 5.

[0102] The expressions “isolate obtainable according to the process of the invention” and “isolate according to the invention” may be used interchangeably.

[0103] The isolate according to the invention has a degree of hydrolysis (or DH) of less than 10%, preferably less than 5%, including DH of 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% and 1%. As well as any range included between two of each of these values.

[0104] The degree of hydrolysis can be determined by measuring the free amino nitrogen content by the OPA method described in the article Nielsen et al., 2001 (Nielsen et al., Journal of Food Science, Volume 66, Issue , Pages 642-646, 2001 “Improved Method for Determining Food Protein Degree of Hydrolysis”) relative to the total nitrogen measured by the DUMAS method according to ISO 16634-2:2016.

[0105] The Hydrolysis Degree Measurement Test described below is particularly preferred. Its principle consists of first determining the amino nitrogen content (free NH2 functions) in the protein sample (preferably with the MEGAZYME kit (reference K-PANOPA), then determining the protein nitrogen content (total nitrogen therefore including free and engaged NH2 functions) of the sample, and finally calculating the degree of hydrolysis accessible via the ratio of these two measurements.

[0106] Determination of amino nitrogen content:

[0107] The "amino nitrogen" groups of the free amino acids in the sample react with N-acetyl-L-cysteine ​​and Ophthaldialdehyde (OPA) to form isoindole derivatives. The amount of isoindole derivative formed during this reaction is stoichiometric with the amount of free amino nitrogen. It is the isoindole derivative that is measured by the increase in absorbance at 340 nm. In a 100 mL beaker, place a precisely weighed test portion P* of the sample to be analyzed. This test portion will be 0.5 to 5.0 g depending on the amino nitrogen content of the sample. Approximately 50 mL of distilled water is added, homogenized, and transferred to a 100 mL volumetric flask. 5 mL of 20% sodium dodecyl sulfate (SDS) is added and the volume is made up to 100 mL with distilled water. Stir for 15 minutes with a magnetic stirrer at 1000 rpm. Solution No. 1 is prepared by dissolving one tablet from bottle 1 of the Megazyme kit in 3 mL of distilled water and stirring until completely dissolved. One tablet is required per test. Solution No. 1 is prepared extemporaneously. A blank, a standard and a sample are prepared directly in the spectrophotometer cells under the following conditions: -blank: introduce 3.00 ml of solution no. 1 and 50 μl of distilled water -standard: introduce 3.00 ml of solution no. 1 and 50 μl of bottle 3 of the Megazyme kit -sample: introduce 3.00 ml of solution no. 1 and 50 μl of the sample preparation. The contents of each tank are mixed and the absorbance measurement (A1) of the solutions is read after approximately 2 minutes using a spectrophotometer at 340 nm (spectrophotometer equipped with 1.0 cm optical path cuvettes, capable of measuring at a wavelength of 340 nm, and verified according to the operating procedure described in the manufacturer's technical manual relating to it). The reactions are then initiated immediately by adding 100 µl of solution no. 2, which corresponds to the OPA solution from bottle 2 of the Megazyme kit, to each spectrophotometer cuvette. The contents of each tank are mixed and placed in the dark for approximately 20 minutes. The absorbance measurement A2 of the blank, the standard and the sample is then read on the spectrophotometer at 340 nm. The free amino nitrogen content, expressed as a percentage by weight relative to the weight of the product, is given by the following formula: [Math.1] AAech - àAblc) x 3.15 x 14.01 x V x 100 % free amino nitrogen = 6803 x 0.05 xmx 1000 [Math.2] (AAech - àAblc) x 12.974 x V % free amino nitrogen = - mx 1000 where: AAech =Aech2 - Aechl AAblc =Ablc2 - Ablc1 Aech2 = absorbance of the sample after addition of solution no. 2 Aechl = absorbance of the sample after addition of solution no. 1 Ablc2 = absorbance of the blank after addition of solution no. 2 Ablcl = absorbance of the blank after addition of solution no. 1 V = volume of the flask m = mass of the test sample in g 6803 = extinction coefficient of the isoindole derivative at 340 nm (in L. mol -1 .cm -1 ). 14.01 = molar mass of nitrogen (in g. mol -1 ) 3.15 = final volume in the tank (in mL) 0.05 = test sample in the tank (in mL)

[0108] Determination of protein nitrogen content:

[0109] The protein nitrogen content is determined according to the DUMAS method according to ISO 16634 - 2016 standard. It is expressed as a percentage by weight relative to the weight of the product.

[0110] Calculation of the degree of hydrolysis: The degree of hydrolysis (DH) is calculated with the following formula: [Math.3] % amino nitrogen DH = - ; - x 100 % protein nitrogen

[0111] The isolate obtainable according to the process of the invention can also be characterized by a reduced content of volatile compounds responsible for undesirable stimuli.

[0112] In one embodiment, the isolate obtainable according to the method of the invention is characterized by a reduced content of volatile compounds responsible for plant flavors, for example herbaceous flavors or pea flavors ("beany" flavors).

[0113] The organic compounds responsible for plant flavors may in particular be compounds comprising one or more aldehyde groups and / or ketones. In the present application, the term “aldehyde” may be used to designate a compound comprising one or more -CHO groups, preferably 1 or 2 -CHO groups, more preferably 1 single -CHO group. The term “ketone” may be used to designate a compound comprising one or more -CO-R groups, preferably one or two -CO-R groups, more preferably a single -CO-R group.

[0114] Aldehydes responsible for plant flavors are, for example, aldehydes with green notes ("green-note aldehydes") such as hexanal, nonanal, 2-nonenal, benzaldehyde, 3-methylbutanal.

[0115] An example of a ketone responsible for plant flavor is 3-octen-2-one.

[0116] Organic compounds responsible for plant flavors can also be alcohols such as 1-octen-3-ol, or pyrazines such as 2,5-dimethylpyrazine.

[0117] In one embodiment, the isolate obtainable according to the method of the invention has a reduced content of at least 1, 2, 3, or 4 volatile compounds responsible for plant flavors chosen from hexanal, nonanal, 2-nonenal, benzaldehyde, 3-methylbutanal, 3-octen-2-one, 1-octen-3-ol, and 2,5-dimethylpyrazine.

[0118] The presence of these compounds, even at low levels, in conventional commercial isolates can give them a so-called "vegetal" flavor, for example a pea flavor which is undesirable when the isolate is used to prepare food products. The method according to the invention preferably makes it possible to obtain an isolate whose content of at least one volatile compound responsible for plant flavor is reduced by more than 50%, preferably by more than 25%, the reduction values ​​potentially being 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14% 13% 12%, 11% 10%, 9%, 8% 7%, 6%, 5%, 4% 3%, 2% or 1%. As well as any range included between two of each of these values.

[0119] In one embodiment, the isolate obtainable by the method of the invention is characterized by a reduced content of compounds comprising one or more thiol groups, in particular a reduced content of methanethiol.

[0120] The synthesis of this compound resulting from the degradation of sulfur amino acids is observed in conventional commercial isolates. Its presence in the order of ppb is sufficient to give a so-called "rotten egg" or "hydrogen sulfide" flavor when the isolate is used to prepare ready-to-drink beverages or wet extrusion strips intended to produce meat analogues. Without being bound by any theory, these processes, by generating significant heat, will cause a significant appearance of degradation compounds derived from methanethiol. The method according to the invention preferably makes it possible to obtain an isolate whose methanethiol content is reduced by more than 30%, 40%, 50%, preferably by more than 50%, the reduction values ​​being potentially 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14% 13% 12%, 11% 10%, 9%, 8% 7%, 6%, 5%, 4% 3%, 2% or 1%.

[0121] In one embodiment, the isolate obtainable by the method of the invention is characterized by both a reduced content of volatile compounds responsible for plant flavors and a reduced content of methanethiol. In the present application, the term "thiol" may be used to designate a compound comprising at least one -SH group, preferably comprising 1 or 2 thiol groups, more preferably comprising a single thiol group.

[0122] The plant protein isolate according to the invention is characterized in that its protein content expressed in relation to its total dry matter is between 80% and 95%, preferably between 82% and 92%, preferably between 84% and 90%, preferably between 84% and 88%. It will therefore be considered that a plant protein isolate according to the invention has a protein content expressed in relation to its total dry matter of 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% or 95%. As well as any range included between two of each of these values.

[0123] The invention also relates to the use of the isolate according to the invention or obtained according to the process of the invention for industrial applications including food, nutraceutical and pharmaceutical applications.

[0124] The human and animal food industry means industrial confectionery (e.g. chocolate, caramel, jelly sweets), bakery products (e.g. bread, brioches, muffins), the meat and fish industry (e.g. sausages, hamburgers, fish nuggets, chicken nuggets), sauces (e.g. Bolognese, mayonnaise), milk products (e.g. cheese, plant-based milk), beverages (e.g. protein-rich drinks, powdered drinks for reconstitution).

[0125] In general, the material rich in vegetable proteins whose organoleptic properties have been improved with the process of the invention, in other words the composition according to the invention, in particular the isolate according to the invention, can be used in food products at a content of up to 100% by weight relative to the total dry weight of the food, for example, of a quantityfrom about 1% by weight to about 80% by weight based on the total dry weight of the food or beverage. All amounts in between (i.e., 2%, 3%, 4%... 77%, 78%, 79% by weight based on the total weight of the food or beverage) are contemplated, as are all intermediate ranges based on these amounts. Food products that may be contemplated in the context of the present invention include baked goods; baked goods (including, but not limited to, rolls, cakes, pies, pastries, and cookies); pre-made sweet bakery mixes for preparing sweet bakery products;Pie fillings and other sweet fillings (including, but not limited to, fruit pie fillings and nut pie fillings such as pecan pie fillings, and fillings for cookies, cakes, pastries, confectionery products and similar products, such as fat-based cream fillings); desserts, gelatins and puddings; frozen desserts (including, but not limited to, frozen dairy desserts such as ice cream - including regular ice cream, soft-serve ice cream and all other types of ice cream - and frozen non-dairy desserts such as non-dairy ice cream, sorbet and similar products); carbonated beverages (including, but not limited to, soft-serve carbonated beverages);non-carbonated beverages (including, but not limited to, soft non-carbonated beverages such as flavored beverages), fruit juices, and sweetened tea or coffee-based beverages); beverage concentrates (including, but not limited to, liquid concentrates and syrups, as well as non-liquid concentrates, such as freeze-dried and / or powdered preparations); yogurts (including, but not limited to, high-fat, reduced-fat, and fat-free dairy yogurts, as well as non-dairy and lactose-free yogurts and frozen equivalents of all these products); snack bars (including, but not limited to, cereal, nut, seed, and / or fruit bars);bread products (including, but not limited to, leavened and unleavened breads, leavened and unleavened breads such as soda breads, breads comprising any type of wheat flour, breads consisting of any type of non-wheat flour (such as potato, rice and rye flour), gluten-free breads); pre-prepared bread mixes for the preparation of bread products; sauces, syrups and dressings; sweetened spreads (including, but not limited to, jellies, jams, butters, nut spreads and other spreadable preserves, preserves and the like); confectionery products (including, but not limited to, jelly beans, soft candies, hard candies, chocolates and gums); sweetened and unsweetened breakfast cereals (including, but not limited to, extruded breakfast cereals, flaked breakfast cereals and puffed breakfast cereals) and cereal coating compositions for use in the preparation of sweetened breakfast cereals. Other types of food and beverage products not mentioned herein but which typically include one or more nutritive sweeteners may also be contemplated within the context of the present invention.In particular, animal feed (such as pet food) is explicitly considered. It can also be used, after extrusion texturization, in meat products such as emulsified sausages or vegetable burgers. It can also be used in egg replacement formulations.

[0126] The composition of the invention can be used as a sole source of protein, but can also be used in combination with other plant or animal proteins. The term "plant protein" refers to all proteins derived from cereals, oilseed plants, legumes and tuberous plants, as well as all proteins derived from algae and microalgae or fungi, used alone or in a mixture, chosen from the same family or from different families. In the present application, the term "cereals" refers to cultivated plants of the grass family producing edible grains, for example wheat, rye, barley, maize, sorghum or rice. Cereals are often ground into flour, but are also supplied as grains and sometimes as whole plants (fodder).In the present application, the term "tubers" covers the storage organs, generally underground, which ensure the survival of plants during the winter and often their multiplication by the vegetative process. These organs are bulbous due to the accumulation of storage substances. The organs transformed into tubers can be the root, e.g. carrot, parsnip, cassava, konjac), the rhizome (e.g. potato, Jerusalem artichoke, Japanese artichoke, sweet potato), the base of the stem. (more specifically the hypocotyl, e.g. kohlrabi, celeriac), the combination of root and hypocotyl (e.g. beetroot, radish). For the purposes of the present invention, the term "legumes" means any plant belonging to the family Cesalpiniaceae, the family Mimosaceae or the family Papilionaceae, and in particular: all plants belonging to the family Papilionaceae, e.g. peas, beans, soybeans, broad beans, green beans, lentils, alfalfa, clover or lupin. This definition includes in particular all plants described in one of the tables in the article by R. Hoover et al., 1991 (Hoover R. (1991) "Composition, structure, functionality and chemical modification of legume starches: a review", Can. J. Physiol. Pharmacol., 69, pp. 79-92). Animal proteins can be, for example, egg or milk proteins, such as whey proteins, casein proteins or caseinate proteins.The pea protein composition can therefore be used in combination with one or more of these proteins or amino acids in order to improve the nutritional properties of the final product, for example to improve the PDCAAS of the protein or to provide other or modify it. [0127JThe invention relates in particular to the use of the isolate according to the invention for the manufacture of biscuits, muffins, pancakes, nutritional bars (intended for specialized nutrition / slimming or sports), breads or gluten-free breads enriched with proteins, small cereals obtained by extrusion cooking ("crisps") with high protein, where high protein solutions are more particularly sought without negative impact on the preparation process or the texture of the preparations or finished products.

[0128] For the purposes of the invention, the term "powdered nutritional formulations" means powdered formulations comprising at least, preferably only, one legume protein, and in particular pea or field bean protein, according to the invention, which can be reconstituted with an aqueous liquid, and which are suitable for oral administration to a human being. [0129JThe term "dry blend" as used herein, unless otherwise indicated, refers to the mixing of components or ingredients to form a base nutritional powder or, the addition of a dry, powdered or granulated component or powder-based ingredient to form a powdered nutritional formulation.

[0130] All percentages, parts and ratios, as described herein, refer to the weight of the total formulation, unless otherwise stated.

[0131] The powdered food formulations and methods of manufacturing the same of the present invention may comprise, consist of, or consist essentially of the essential elements of the invention as described herein, as well as any additional or optional elements described herein or otherwise useful in the nutritional formulation applications.

[0132] The powdered nutritional formulations of the present invention are generally in the form of free-flowing or substantially free-flowing particulate compositions, or at least particulate compositions that can be readily molded and measured using a spoon or other similar device, wherein the compositions can readily be reconstituted by the intended user with an aqueous solution, typically water, to form a liquid nutritional formulation for immediate oral or enteral use. In this context, use "immediately" generally means within about 48 hours, more typically within about 24 hours, preferably immediately after reconstitution.

[0133] Powdered food formulations can be formulated with all types and quantities of nutrients sufficient to form a food supplement, or a specialized nutritional formulation intended for use by people following a specific diet for sports and weight loss.

[0134] In exemplary embodiments, the powdered nutritional formulation may be formulated for use: to repair muscles after intense exercise, for example in athletes, - to ensure the maintenance or construction of muscle mass in athletes, or - as a meal replacement for people wishing to lose weight via a satiating effect.

[0135] Powdered food formulations can have a caloric density tailored to the nutritional needs of the end user, although in most In this case, reconstituted powders contain about 350 to about 400 kcal / 100 ml.

[0136] Powdered food formulations may have a protein level tailored to the nutritional needs of the end user, although in most cases reconstituted powders comprise from about 20 to about 91 g of protein / 100 g, including from about 40 to about 65 g of protein / 100 g.

[0137] Thus, the formulation may comprise between 20 and 95% protein relative to the total weight of the formulation, for example between 20-90%, 30-80%, or 40-60%.

[0138] For example, the legume protein isolate, preferably pea or faba bean, according to the present invention may represent 40-50%, 50-60%, 60-70%, 70-80%, 80-90% or 90-100% of the total protein in the formulation, or any combination of these percentage ranges. 100% represents the ultimate preferred mode in order to maximize the FGF19 overexpression effect.

[0139] Furthermore, powdered food formulations may have a fat content adapted to the nutritional needs of the end user, although in most cases, reconstituted powders comprise from about 0.5 to about 13 g / 100 g, including from about 3 to about 7 g / 100 g. Thus, the formulation may comprise between 0 and 20% of lipids relative to the total weight of the formulation, for example between 0.5-15%, 1-10%, or 3-7% (in particular % by weight).

[0140] The powdered nutritional formulations of the present invention may be packaged and sealed in single or multi-use containers and then stored at ambient conditions for up to about 36 months or longer, more typically about 12 to about 24 months.

[0141] For multi-use containers, they can be opened and covered for repeated use by the end user, provided the covered package is then stored at ambient conditions (e.g., avoid extreme temperatures) and the contents used within approximately a month or two.

[0142] The fields of application of the nutritional formulations according to the invention are in particular: - dietary nutrition (sport, slimming), - clinical nutrition (in the form of a drink, dessert cream or enteral bag), - dairy products (in the form of yogurts, dairy drinks, dairy creams, frozen desserts or sorbets). - biscuit products, pastry products, bread products and high-protein cereal products.

[0143] In the sports world, it is well known that protein contributes to muscle maintenance and growth. Protein intake is also important for athletes who practice bodybuilding or muscle strengthening.

[0144] These proteins must be balanced in terms of amino acid profile and must comply with FAO / WHO recommendations. Their digestibility is an important factor, ranging from rapid to slower digestibility depending on the timing of protein intake.

[0145] Ready-to-drink protein or high-protein drinks provide the body with a choice protein intake, without the calories.

[0146] These high protein drinks must: - be rich in protein, low in carbohydrates and fats; - have good taste; - be designed to help with weight loss, by stimulating fat loss and aiding muscle recovery; - be satietogenic; - help you cope with cravings, without added sugars or fats; - present a balanced content of essential amino acids, fibers, vitamins and minerals; - be low-calorie.

[0147] These ready-to-drink beverages can advantageously be prepared with legume protein isolates, preferably from fava beans or peas, in accordance with the invention. They can also be used as the sole source of protein, preferably because they maximize the effect of overexpression of FGF19.

[0148] For example, plant-based drinks that are alternatives to cow's milk contain on average 4.5 to 11 g of protein per 100 ml of drink, preferably around 7 g of protein per 100 ml, and are very low in fiber (around 0.5 to 1 g per 100 ml).

[0149] Thus, the drink can include between 1 and 20% of protein relative to the total weight of the drink, for example between 3-15%, or 6-8%.

[0150] For example, the pea protein isolate of the present invention may represent 50-60%, 60-70%, 70-80%, 80-90% or 90-100% of the total protein, or any combination of these percentage ranges. Preferably, it represents at least 52%. In particular, the pea protein content is between 52 and 100% of the total protein content.

[0151] For ready-to-drink beverages, the pea protein content may range from 0 to 100%, preferably from 0.01 or 0.1 to 100%. For example, the pea protein isolate of the present invention may represent 0.1-10%, 10-20%, 20-30%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90% or 90-100% of the total protein, or any combination of these percentage ranges.

[0152] In the field of "slimming" drinks, i.e. intended for use in low-calorie diets or for weight loss, as mentioned above, these protein or protein-enriched drinks are not only effective for rapid muscle gain. This type of drink is also very beneficial as part of a slimming diet based on protein consumption.

[0153] It is well known that slimming drinks are ideal for aiding weight loss. In particular, they help to: - provide a satiety effect - protect muscles and tone the body, preventing weight gain.

[0154] As with “sports” drinks, these slimming drinks have: - a balanced content of essential amino acids, fiber, vitamins and minerals - reduced sugar, fat and calorie content.

[0155] This is why protein drinks are so effective for losing a few pounds quickly. These protein-rich preparations simply reduce or stop the feeling of hunger in the person who consumes them. By taking such a drink, for example, the user can significantly reduce the amount of food consumed, and allow for rapid weight loss (as part of a meal replacement process for weight control, or replacement of the total daily ration for weight control).

[0156] In clinical nutrition, it is known that enteral nutrition is a therapeutic solution of tube nutrition which is used when the digestive tract is functional and accessible but when the patient cannot eat normally or in cases of severe malnutrition.

[0157] This technique allows nutrients to be delivered directly into the digestive tract. It replaces, in whole or in part, traditional oral feeding with "complete" nutritional formulas providing all the nutrients necessary for the body.

[0158] These formulas are generally packaged in flexible bags (PVC) and administered by means of nasogastric tubes or gastrostomies, nasojejunal, nasoduodenal, jejunostomy.

[0159] These nutritional blends are composed of proteins, lipids, carbohydrates, vitamins and minerals with or without fiber.

[0160] There are several categories: polymeric mixtures (standard) and semi-elemental mixtures ("predigested"), the latter being indicated in very specific cases (short bowel syndrome, exocrine pancreatic insufficiency, etc.): - Polymeric blends - low-calorie (0.5 - 0.75 kcal / ml), normal or high-protein, with or without fiber - isocaloric (1 kcal / ml), normal or high protein, with or without fiber - high-calorie (1.25-1.5 kcal / ml) normal or high-protein, with or without fiber - specific formulas (glycemic metabolism disorders, respiratory failure).

[0161] Semi-elementals are iso or hypercaloric, normo or hyperprotein mixtures, based on peptides and medium-chain triglycerides.

[0162] Pea protein isolates, as a source of protein, due to their functional properties are particularly well suited for this use.

[0163] Furthermore, they allow the same properties to be preserved as milk proteins, and at a lower cost.

[0164] The invention will be better understood with the following examples which are only intended to make it better understood. These have no limiting scope. Brief description of the drawings

[0165] Other characteristics, details and advantages of the invention will appear on reading and analyzing the attached drawings, in which:

[0166] [Fig 1] illustrates the impact of strain selection among Limosilactobacillus fermentum, Limosilactobacillus Reuteri, Limosilactobacillus Rhamnosus and Lacticaseibacillus rhamnosus (Lr) after contact with Nutralys® F85F on their efficiency in modifying volatile compounds.

[0167] [Fig 2] illustrates the impact of the culture time of Limosilactobacillus fermentum (Lf) and Lacticaseibacillus rhamnosus (Lr) strains before contact with Nutralys® F85F on their effectiveness in improving organoleptic qualities. F85F = Nutralys® F85F; B = blank; Lf 5h = Lactobacillus fermentum pre-cultured for 5h; Lf 10h = Lactobacillus fermentum pre-cultured for 10h; Lf 15h = Lactobacillus fermentum pre-cultured for 15h; Lr 5h = Lactobacillus rhamnosus pre-cultured for 5h; Lr 10h = Lactobacillus rhamnosus pre-cultured for 10h; Lr 15h = Lactobacillus rhamnosus pre-cultured for 5 to 15 h.

[0168] [Fig 3] illustrates the impact of sugar composition on the performance of the Limosilactobacillus fermentum strain in modifying the profile of volatile compounds present in a sample.

[0169] [Fig 4] illustrates the proportional distribution of the different volatile compounds grouped by sensory profile in the raw Nutralys® pea isolate and fermented by the different strains in Example 1. Examples

[0170] Example 1: Process according to the invention starting from a pea isolate

[0171] Strains and culture conditions: - From a cryotube (frozen strain), a 10 mL pre-culture tube of the medium appropriate to the strain is inoculated with a dose. - The tube is incubated in a non-agitated incubator, at the optimal temperature for each strain for 15 to 24 hours. - The pre-culture tube is then used to inoculate a 1L flask or Erlenmeyer flask with 1% medium. - Propagation takes place at the optimal temperature for each strain during the optimal time for each. - The growth parameters of the strains are summarized in Table 2 below: [Table 2]

[0172] (MRS = De Man Rogosa and Sharpe agar; MW = Malt Wickerham agar)

[0173] Preparation of the inoculum: - At the end of the growth period, a wash is applied to the different cultures in order to remove the culture medium. - Cultures are centrifuged at 4000 G, at 4 °C for 10 min. The supernatant is removed and the pellet is resuspended in physiological saline. - A new centrifugation is then carried out and the supernatant removed. - The strains are then resuspended in a volume of physiological water, depending on the calculated biomass, in order to be able to inoculate the test medium at 10 8 CFU / mL (so in order to have an inoculum at 10 9 CFU / mL).

[0174] Preparation of the matrix (test medium): - The test medium or matrix consists of a 10% solution of pea isolate (Nutralys® S85F). 100 g of the pea isolate are introduced into sterile distilled water in a thin stream, with stirring, in order to obtain 900 mL of suspension. - 100 mL of inoculum will be added to this volume to obtain 1 L of suspension. - The suspension is left stirring for 30 minutes to ensure good hydration and solubilization of the protein isolate.

[0175] Whole cell biomass treatment (abbreviated WCB. Can also be called “whole cell biocatalysis”): Action of strains on the test medium with a short contact time (the principle being to use the cell as a metabolic catalysis system, with little or no cell growth): - After preparing a 10 inoculum 9 CFU / mL, the test medium is inoculated with 100 mL of inoculum in 900 mL of test matrix. - Once the medium is inoculated, the Pyrex Erlenmeyer flasks with stoppers are placed in a stirring oven at 130 rpm at 30°C for one hour. - After treatment, the sample is spray-dried to stop the action of the strains. Spray-drying is carried out at 160°C, with 10% product feed and 100% suction. - The sample collected in powder form is stored in a sterile glass bottle, until the volatile compounds are measured by SPME-GC-MS.

[0176] WCB Results - Interpretations The results are presented in Figure 1. The impact on volatile compounds is presented as area per g of sample (i.e., pea isolate), average of three samples. The results shown in Figure 1 are grouped by chemical family type.

[0177] The results show that the method of the invention makes it possible to modify the volatile compound profile of the treated pea isolate (Nutralys® S85F). The conversion of aldehydes into alcohols is very high in obligate heterofermentatives (Limosilactobacillus fermentum and Limosilactobacillus reuteri). Limosilactobacillus reuteri is also effective on ketones. The action is also visible in Saccharomyces cerevisiae. In Lacticaseibacillus rhamnosus, the conversion of aldehydes to carboxylic acids is also observed (not detected).

[0178] The conversion of aldehydes and ketones into other compounds (alcohols and / or carboxylic acids) results in a reduction of the undesirable "beany" or "vegetal" flavor. Indeed, aldehydes and ketones are the main contributors and the products of their bioconversions have less characteristic odors with higher perception thresholds. These undesirable flavors are therefore less or even no longer perceived in the product. The reduction in the quantity of green-note aldehydes leads to an organoleptic improvement of the products, by a reduction of the "vegetal" sensory attributes.

[0179] Chemical analyses are carried out and presented in Table 8 below:

[0180] We therefore note that with the method according to the invention: - The production of lactic acid and ethanol is very low. - pH and viability do not vary, proving an absence of growth. - The DH does not vary, demonstrating an absence of hydrolysis.

[0181] A panel of 5 people tasted the different samples: - Nutralys® S85F pea isolate is described as bitter and plant-based - Treated with Lacticaseibacillus rhamnosus LGG, the vegetal note disappears benefit from a milky note. Such a note allows us to consider an interest in milk-like drinks - Treated with Limosilactobacillus fermentum, the vegetal note disappears in favor of an umami note. - Treated with Saccharomyces cerevisiae, the vegetal note disappears in favor of a cereal note. - Treated with Gluconobacter suboxydans, the vegetal note disappears but leaves no room for any other flavor of interest.

[0182] Figure 4 shows the proportional distribution of different volatile compounds within different samples generated using Example 1, grouped by sensory profile.

[0183] The grouping by sensory profile is carried out on the basis of literature, in particular the website www.thegoodscentscompany.com.

[0184] Thus, the groupings of volatile compounds are as follows: - The “Green” group will be composed of Hexanal, Heptanal, 2-Hexenal, 2- Heptenal, (E)-, 2,4-Heptadienal, (E,E)-, 2,6-Nonadienal, (E,Z)-, 1- Heptanol,2-Octen-1-ol, (E)-, 3-Heptanone, Furan, 2-pentyl-, 1-Octanol, 2,4- Octadienal, (E,E)- and 2-Pentenal, (E)-. - The “Bold” group will be composed of 2-Octenal, (E)-, 2-Nonenal, (E)- and (Z)-, 2-Decenal, (E)- and (Z)-, 2-Nonen-1-ol, (E)- and 3,5-Octadien-2-one, (E,E)-. - The “Herbal” group will consist of 2-Dodecenal, 1-Hexanol, 3-Octanone and Eucalyptol - The “Others” group will be composed of 2,3-Octanedione, Octanoic acid and methyl ester - The “Earth” group will be composed of 1-Octen-3-ol, 2-Octanone, 1-Octen- 3-one and 3-Octen-2-one, (E)- - The “Bitter Almond” group will be composed of Benzaldehyde. - The “Aldehydic” group will be composed of Octanal, Nonanal and Decanal - The “Roasted Chocolate” group will be composed of Butanal and 3-methyl-Furan, 2-(1-pentenyl)-, (E)- - The “Fermented” group will be composed of Pentanal, 1-Pentanol, Decanoic acid, methyl ester - The “Sulfurous” group will be composed of Disulfide and Dimethyl trisulfide - The “Fruity / Floral” group will be composed of 2-Pentanone, 1-Nonanol, 1 - Hexanol, 2-ethyl-, 2-Nonanone, 2-Decanone, 5-Hepten-2-one, 6-methyl-, 2- n-Butyl furan, 2(3H)-Furanone, 5-butyldihydro, 2(3H)-Furanone, 5- hexyldihydro-, Phenylethyl Alcohol, Hexanoic acid, methyl ester, 3-Nonen-2- one, 2-Undecanone - The “Cheese” group will be composed of 2-Heptanone - The “Solvent / Alcohol” group will be composed of 2-Propanone, Acetaldehyde, Ethanol and Furan, 2-ethyl-

[0185] Figure 4 is interpreted as follows: - The profiles of Nutralys (untreated pea isolate) and Nutralys treated with G. suboxydans are very similar, with approximately % of compounds from the “Green” group. While the content of these compounds decreases, the overall sensory profile remains the same. - The profiles of Nutralys treated with S. cerevisiae and L. fermentum are very similar, with only about 50% of compounds from the “Green” group. The remainder is therefore made up of volatile compounds from other groups, providing a more complex profile. - The profiles of Nutralys treated with L. reuteri and L. rhamnosus are similar, with a total disappearance of volatile compounds from the “Green” group. Their sensory profiles are radically different.

[0186] In conclusion and in comparison with the patent EP 0 255 588 teaching the use of Gluconobacter suboxydans, our process does not just reduce the vegetal note. The process according to the invention allows this to be done while creating complex and unique organoleptic profiles.

[0187] Due to the reduced contact time, no lactic acid production takes place. The protein is not modified, especially the degree of hydrolysis. [01881 Example 2: Process outside the invention, using the strains of interest but using them in fermentation:

[0189] The purpose of this example is to demonstrate that a long contact time between the inoculum and the isolate, typically 8 hours or 24 hours according to the publications of the prior art, as described mainly in the prior art with these strains does not make it possible to obtain the same composition as according to the invention.

[0190] Obtaining inocula: Medium : - MRS (ready to use, Merck) for the strains Limosilactobacillus fermentum, Limosilactobacillus reuteri, and Lacticaseibacillus rhamnosus. - YE glc (Bacto Yeast Extract, Difco 10g / L + glucose 40g / L) for Saccharomyces cerevisiae and Gluconobacter suboxydans strains. Culture: o In a 500mL unbaffled Erlenmeyer flask containing 100mL of medium for the strains Limosilactobacillus fermentum, Limosilactobacillus reuteri, and Lacticaseibacillus rhamnosus, In a 4-baffle Erlenmeyer flask at the bottom + 1 drop of antifoam containing 100mL of medium for the strains Saccharomyces cerevisiae and Gluconobacter suboxydans. o Inoculation: 1% with a frozen stock tube (1 mL) o Incubation: Anaerobically (bell) for 16h at 37°C, 80rpm Limosilactobacillus fermentum, Limosilactobacillus reuteri, and Lacticaseibacillus rhamnosus. Aerobic for 16 hours at 150 rpm, 30°C for Gluconobacter suboxydans and 37°C for Saccharomyces cerevisiae o Biomass preparation: Centrifuge the biomass from the Erlenmeyer flasks at 3000g for 10 minutes for all strains except Gluconobacter suboxydans, which will require centrifugation at an acceleration of 10000g. Remove the supernatant and recover the biomass with 10mL of sterile physiological saline at 4°C. (Washing the biomass is not necessary given the inoculum required). Measure the OD at 600nm and count the cell suspensions. Fermentation of Nutralys® S85F 10% at native pH: - Equipment 2L unbaffled Erlenmeyer flasks x11 Sterile demineralized water bottles; approx. 6L - Procedure: o Pre-incubate the water bottles at 30 and 37°C at 110rpm. o Place 50g of Nutralys® S85F in each Erlenmeyer flask o Add sterile water qsp 500g o Leave the Nutralys® to rehydrate for 30 minutes at the incubation temperature of the different tests. - Erlenmeyer flask incubation parameters o Nutralys: Nutralys + Gluconobacter oxydans: 30°C 110rpm o Nutralys + Saccharomyces Cerevisiae: 37°C 110rpm o Nutralys + Limosilactobacillus fermentum, Limosilactobacillus reuteri, and Lacticaseibacillus rhamnosus: 37°C 60rpm + anaerobic bell - Calculate the volume of inoculum to achieve an initial viability of 1.10 7 CFU / mL Correlations between DO and viability (symbolized by "V0"): ■ Saccharomyce cerevisiae CBS 8066: D.0.600nm = 1VO 1,1.10 7 CFU / mL ■ Gluconobacter suboxydans ATCC 19357 (or ATCC 23773): D.0.600nm = 1VO 2, 5.10 8 CFU / mL ■ Lacticaseibacillus rhamnosus GG LMG 18243 (LGG): D.0.600nm = 1VO 6.8.10 7 CFU / mL ■ Limosilactobacillus fermentum CNCM I-5802: D.0.600nm = 1VO 3.0.10 8 CFU / mL ■ Limosilactobacillus reuteri DSM 17509: D.0.600nm 1 Uo 5.0.10 7 CFU / mL o Double the Nutralys® Erlenmeyers for each strain (2 x 5 strains + 1 Control) o Inoculate all the tests as early as possible in the morning o Take a sample of approximately 5 mL at tO on 1 of the 2 Erlenmeyers o Carry out the following analyses: pH, Ethanol (Cedex Bio), Lactate (Cedex Bio), Viability (inocula + tO), Acetic acid, DH, Protein content (N6.25), Dry matter. o Stop the Control Erlenmeyer and transfer it to a flask for freezing. o After 8 hours of fermentation, stop one of the 2 Erlenmeyers for each strain tested. o Carry out the following analyses: pH, Ethanol (Cedex Bio), Lactate (Cedex Bio), Viability (inocula + tO), Acetic acid, DH, Protein content (N6.25), Matter dry. o After 24 hours of fermentation, stop the last Erlenmeyer for each strain tested. Carry out the following analyses: pH, Ethanol (Cedex Bio), Lactate (Cedex Bio), Viability (inocula + tO), Acetic acid, DH, Protein content (N6.25), Dry matter.

[0191] The results are presented in Table 3 and Table 4: [Table 3] 0192]Table 3: Ethanol / lactate / pH / viability measurements [Table 4] 0193]Table 4: MS / DH / Nx6.25 measurements

[0194] In conclusion, the use of the strains of interest in a process outside the invention (long contact time) produces an isolate containing high levels of lactic acid. The DH in particular for L. reuteri, S. cerevisae and G. suboxydans increases, a sign of protein hydrolysis.

[0195] Example 3: Demonstration of the importance of pre-WCB culture time:

[0196] The same protocol as for example 1 is applied. However, the tests are carried out on a volume of 250 mL, with 25 g of Nutralys®.

[0197] The impact of strain propagation time on their effectiveness as biocatalysts is studied in this example. Three propagation times for inoculum preparation are tested: cells are harvested after 5h, 10h or after 15h.

[0198] Two strains are tested: Limosilactobacillus fermentum (Lf) and Lacticaseibacillus rhamnosus (Lr .

[0199] Results - Interpretations

[0200] The results are presented in Figure 2.

[0201] Figure 2 illustrates the impact of the culture time of Limosilactobacillus fermentum (Lf) and Lacticaseibacillus rhamnosus (Lr) strains before contact with Nutralys® F85F on their effectiveness in improving organoleptic qualities.

[0202] Two cases are observed: - For Limosilactobacillus fermentum (obligate heterofermentative), the minimum propagation time to obtain the best biocatalysis activity is 10 hours. - For Lacticaseibacillus rhamnosus (facultative heterofermentative), the minimum propagation time is 15 hours.

[0203] Different cultivation times are therefore necessary depending on the strains to obtain a maximum effect. A minimum propagation time of 10 hours is preferable to obtain the maximum technical effect of the invention.

[0204] Example 4: Demonstration of the importance of sugar concentration during the WCB step:

[0205] In this example, we use the same protocol as in example 1. The interest here is to study the comparison of the impact: - of a chosen strain (Limosilactobacilus fermentum), - on a model matrix (volatile compounds responsible for unwanted flavors added with buffers) replacing pea protein isolate (Nutralys® S85F) - with and without sugars.

[0206] Comparison of three sugar contents: - Og / L, - 0.5 g / L (Nutralys® sugar content in 10% solution, “during the process) - 5 g / L (Nutralys® sugar content).

[0207] A solution at 1.6 ppm of volatile compounds is made, with: - hexanal, - nonanal, - 2-nonenal, - benzaldeyhde, - 3-methylbutanal, - 1-octen-3-ol, -3-octen-2-one and - 2,5-dimethylpyrazine.

[0208] From this solution, three solutions with three sugar levels are prepared, as shown below. - Tracers 1.6 ppm without sugars -Add phosphate buffer (pH 6.5 0.1 M) K2HPO4 4.9 g / L and KH2PO4 9.9 g / L - 6 tubes of 9 mL - Sterilizing filtration (0.22 pm) - Tracers 1.6ppm with 0.5p of sugars - Addition of phosphate buffer (pH 6.5 0.1 M) K2HPO4 4.9 g / L and KH2PO4 9.9 g / L - Adding sugars: [Table 5] ° Sterilizing filtration (0.22 pm)

[0209] Tracers 1.6 ppm with 5q of sugars - Addition of phosphate buffer (pH 6.5 0.1 M) K2HPO4 4.9 g / L and KH2PO4 9.9 g / L - Addition of 5g of sugars: [Table 6] - 6 tubes of 9 mL - Sterilizing filtration (0.22 pm) [0210lContact - The same protocol as for the WCB approach is applied. [02111 Results and Interpretations

[0212] The tests were carried out in three replicates, using three different pre-cultures. The impact of Limosilactobacilus fermentum on the tracers as a function of sugar levels is shown in the figures below.

[0213] The results are presented in Figure 3 and Table 7. [Table 7] on the performance of the Limosilactobacillus fermentum strain in modifying the profile of volatile compounds present in a sample.

[0215] Example 5: Demonstration of the importance of cell concentration before treatment and the possibility of using a freeze-dried inoculum:

[0216] In this example we use the same protocol as in example 1 with the exception of 3 points: - Two strains are studied Limosilactobacillus fermentum and Lacticaseibacillus rhamnosus. - In addition to a frozen strain, a lyophilized strain is introduced. Before its use, it is dispersed for 30 minutes at 30°C in a solution of demineralized water with glucose at 1 g / L and a pea isolate (such as Nutralys® S85F) at 10 g / L. The inoculum is then used as in example 1, except for their concentrations (see below). - After the strains have been resuspended in a volume of water, the necessary quantity is introduced in order to have an inoculum of 10 8 , 10 7 or 10 4 CFU / mL).

[0217] The following facts are observed: - The results obtained with 10 4 CFU / mL are very poor: aldehydes, especially hexanal, are not consumed. The protein composition is therefore unchanged from a flavor point of view. - The results obtained with 10 8 and 10 7CFU / mL are equally good from this point of view, whether with a fresh, frozen or freeze-dried strain. Freeze-drying is therefore an excellent means of preserving strains before implementation.

Claims

Claims

1. A method for improving the organoleptic properties of a material rich in vegetable proteins comprising the following steps:

1. suspending a material rich in vegetable proteins in a preferably aqueous solvent so as to obtain a suspension having a dry matter content of between 5% and 20%, preferably between 7.5% and 15%, even more preferably between 8% and 12%; 2. the addition of one or more microbial strains to the suspension from step 1, the microbial strain(s) being chosen from strains of lactic acid bacteria, strains of yeast, or a mixture thereof, the strain(s) preferably being one or more strains of lactic acid bacteria, so as to obtain a seeded suspension having a cell density of between 1.10 5 and 1.10 10 cfu / mL, preferably between 1.10 7and 1.10 9 cfu / mL, preferably between 5.10 7 and 5.10 8 cfu / mL; 3. incubation of the suspension from step 2 at a temperature between 20°C and 40°C for a time between 30 and 120 minutes, preferably between 45 and 75 minutes, even more preferably between 50 and 70 minutes.

2. Method according to claim 1 characterized in that the material rich in vegetable proteins from step 1 is in the form of a powder containing a quantity of vegetable protein greater than 1% by weight, preferably between 70% and 90% by weight, relative to the weight of powder.

3. Method according to any one of claims 1 or 2, characterized in that the material rich in vegetable proteins from step 1 is an isolate or a concentrate of vegetable proteins, preferably is an isolate of vegetable proteins whose protein content is greater than 70% by dry weight, preferably between 75% and 95% by dry weight, relative to the dry weight of isolate.

4. Method according to any one of claims 1 to 3 characterized in that the material rich in vegetable protein from step 1 comes from of a legume, preferably of a legume selected from the group consisting of peas and field beans, more preferably is derived from peas.

5. Method according to any one of claims 1 to 4, characterized in that the pH of the suspension of material rich in vegetable proteins from step 1 is rectified so as to be between 5.0 and 7.5, preferably between 6.5 and 7.5; preferably so as to be equal to 7.0, before implementing step 2.

6. Method according to claims 1 to 5 characterized in that the sugar content of the suspension of material rich in vegetable proteins from step 1 is rectified so as to contain between 1% and 10% of sugars, preferably between 2% and 8%, even more preferably between 3% and 6%.

7. Method according to claims 1 to 6, characterized in that the strain(s) of step 2 are selected from a strain of Limosilactobacillus fermentum, Limosilactobacillus reuteri, Lactobacillus rhamnosus or Saccharomyces cerevisiae, preferably Limosilactobacillus fermentum, Limosilactobacillus reuteri, or Lactobacillus rhamnosus.

8. Method according to claims 1 to 7 characterized in that the strain(s) used during step 2 have been cultivated according to a method comprising: i. inoculation of a solid culture medium, for example agar, with a sample of the strain(s); ii. pre-cultivation of the strain(s), by incubation of the inoculated solid culture medium from step 1 in a non-stirred incubator, preferably for between 10h and 24h; iii. propagation of the strain, by inoculation of a liquid culture medium with a sample of pre-cultivated strain from step ii, and cultivation of the strain in liquid medium.

9. Method according to claim 8 characterized in that the incubation time of step 11 is between 10h and 20h.

10. Method according to claims 1 to 9 characterized in that step 3 is followed by a step 4 comprising the following sub-steps: 4a. optionally, rectification of the pH of the suspension obtained at the end of step 3 to a pH between 5 and 9, preferably between 6 and 9, preferably a pH of 7, preferably by adding soda or lime to said suspension; 4b. heat treatment of the suspension from step 3 or step 4a between 100°C and 160°C for 0.1 to 1 s, and 4c. drying the suspension from step 4b using an atomizer until a vegetable protein isolate is obtained, preferably having a dry matter content greater than 95% relative to the mass of isolate.

11. A plant protein isolate obtainable by the process of any one of claims 1 to 10.

12. Plant protein isolate according to claim 11, characterized in that its degree of hydrolysis is less than 10%, preferably less than 5.

13. Vegetable protein isolate according to any one of claims 11 to 12, characterized in that its protein content is between 70% and 95% by weight, preferably between 82% and 92% by weight, preferably between 84% and 90% by weight, preferably between 84% and 90% by weight, relative to the total dry matter weight of the isolate.

14. Use of an isolate according to any one of claims 11 to 13 or obtained according to the process of any one of claims 1 to 10 for industrial applications including food, nutraceutical and pharmaceutical applications.

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