Improved plant protein isolates by whole cell biomass processing

Abstract

The invention relates to a method for improving the organoleptic properties of a vegetable protein-rich material, comprising the following steps: 1. Suspending the vegetable protein-rich material in a preferentially aqueous solvent in order to obtain a suspension having a dry matter content of between 5% and 20%; 2. Adding to the suspension of step 1 one or more microbial strains, where the one or more microbial strains are selected from the group consisting of lactic acid bacteria strains, yeast strains or mixtures thereof, so as to obtain an inoculated suspension having a cell density of between 1.105 cfu / mL and 1.1010 cfu / mL; 3. Incubating the suspension of step 2 at a temperature of between 20 DEG C and 40 DEG C for a duration of between 30 minutes and 120 minutes. The invention also relates to a vegetable protein isolate obtainable by the method according to the invention and to the use of the vegetable protein isolate.

Description

Technical Field

[0001] The present invention relates to the field of plant proteins, in particular to the field of leguminous plant protein isolates, and even more particularly to the field of pea protein isolates. Existing technology

[0002] Humans require 12 to 20% of their daily protein intake, which is supplied by both animal-derived (meat, fish, eggs, dairy products) and plant-derived (cereals, legumes, algae).

[0003] However, in developed countries, protein intake is primarily in the form of protein from animal sources. However, numerous studies have shown that excessive consumption of animal-based protein at the expense of plant-based protein is one of the reasons for the increase in cancer and cardiovascular disease.

[0004] Furthermore, animal proteins have numerous disadvantages with regard to their allergenic potential (especially with regard to proteins from milk or eggs) and environmentally related to the harmful effects of intensive farming.

[0005] Consequently, there is an increasing demand from manufacturers for plant-derived compounds that possess beneficial nutritional and functional properties without the drawbacks of animal-derived compounds.

[0006] Soybeans have been and remain the leading plant-based alternative to animal protein. However, the use of soy presents certain disadvantages. The source of soy seeds is often from GMOs, and the production of their protein is carried out via a de-oiling step using solvents.

[0007] Since the 1970s, the development of legumes (including peas in particular) as an alternative protein source to animal protein for animal and human food consumption has increased dramatically in Europe, and primarily in France. Peas contain approximately 27% protein by weight. The term "pea" is used herein in its most widely accepted sense and specifically includes all wild varieties of "smooth peas" as well as all mutant varieties of "smooth peas" and "wrinkled peas," regardless of the intended use (human food, animal feed, and / or other uses) of the variety. These seeds are non-GMO and do not require a de-oiling step using solvents.

[0008] Pea proteins (primarily vicilin) ​​have been extracted and utilized industrially for many years. Several methods exist, which can be divided into two main categories: "dry" and "wet" methods. The former involves reducing the particle size of leguminous seeds to a powder, followed by particle size separation using an updraft, a process commonly known as turbo separation. These methods produce protein concentrates with a protein content not exceeding 60%-70%. These methods are not considered in the context of the present invention, which is concerned with isolates having a protein content greater than these levels.

[0009] Regarding the second category of "wet" methods, EP 1 400 537 can be cited as an example of a method for extracting pea protein. In this method, the seeds are ground in the absence of water (a process known as "dry grinding") to obtain a powder. This powder is then suspended in water to extract the protein by precipitation. Precipitation is achieved by adjusting the isoelectric pH and / or by heating the medium.

[0010] Such methods have a number of disadvantages, notably the production of undesirable vegetal flavors.

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

[0012] First of all, we can mention the use of thermal procedures during the extraction process and / or on the final product. This thermal treatment will eliminate the endogenous enzymes (such as lipoxygenase) that are responsible for the appearance of these undesirable flavors and / or the organic volatile compounds (such as hexanal) that carry these flavors. However, these thermal treatments, which are well known, for example, in patent applications WO 2020 / 260841 or WO 2020 / 064822, have many disadvantages, such as high process costs (due to the acquisition and maintenance of suitable equipment and its use), or the denaturation of proteins that may be sensitive to the thermal treatment.

[0013] The second approach involves adding masking or flavoring compounds to the protein once it is obtained, either during the protein's manufacturing process or when it is primarily formulated into food. Again, many issues persist here, not least the added cost of using such compounds or the regulatory issues involved in using compounds for these purposes.

[0014] Alternative methods consist of using bacterial strains to regulate organoleptic characteristics. During the extraction process (preferentially at the end of the process, i.e. in the case of the final product), bacterial strains are added and subjected to physicochemical conditions that allow them to grow. During this growth period, bacterial strains activate their metabolism to consume several compounds, including those responsible for local flavor and organoleptic characteristics (e.g., hexanal). This paper also continues to have problems, such as the hydrolysis of proteins by proteases that constitute bacterial strains or are produced by bacterial strains, or the generation of unwanted local flavors, such as due to the production of organic acids such as lactic acid.

[0015] Finally, patent application EP 0 255 588 may be mentioned, which describes the use of acetic acid bacteria to reduce the hexanal content and, therefore, the associated vegetal flavors in foods, particularly alcoholic beverages. Example 1 demonstrates the use of the acetic acid bacteria Gluconobacter suboxydans on soy milk. A reduction in hexanal was noted, and a sensory panel confirmed a reduction in the "grassy" flavor. It should be noted that other flavors remain unchanged, so other flavors typically found in plant proteins, such as bitterness or sourness, remain unchanged and may even be enhanced by the absence of hexanal.

[0016] The applicant is commendable for having worked in this field and for having discovered a group of strains that improve the organoleptic properties of plant proteins, and for having developed a method for using this group of strains, with the aim of obtaining protein isolates whose functional and organoleptic properties are optimized to a level not previously achieved. Furthermore, the strains according to the invention and the method for using said strains make it possible to obtain plant protein isolates whose organoleptic properties are of clear interest for the agri-food industry.

[0017] The present invention will be better understood in the following description portion of the present application.

[0018] General Description

[0019] The present invention relates to a method for improving the organoleptic properties of a material rich in vegetable protein, the method comprising the following steps:

[0020] 1. suspending a material rich in vegetable protein 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%;

[0021] 2. Add one or more microbial strains to the suspension of step 1 in order to obtain a suspension having the 5 cfu / mL and 1.10 10 cfu / mL, preferably 1.10 7 cfu / mL and 1.10 9 cfu / mL, preferably between 5.10 7 cfu / mL and 5.10 8 cfu / mL, the microbial strain is selected from a lactic acid bacteria strain, a yeast strain or a mixture thereof, the strain is preferably one or more lactic acid bacteria strains;

[0022] 3. Incubate the suspension of step 2 at a temperature between 20°C and 40°C for a duration between 30 and 120 minutes, preferentially between 45 and 75 minutes, even more preferentially between 50 and 70 minutes.

[0023] In a preferred embodiment, the method according to the invention is characterized in that the vegetable protein-rich material of step 1 is in the form of a powder containing vegetable protein in an amount greater than 1% by weight, preferably between 70% and 90% by weight, relative to the weight of the powder. In this embodiment, the powder is preferably derived essentially from seeds, typically leguminous plant seeds, such as pea or bean seeds, which have been ground and reduced to a powder form. In other words, the powder is preferably a meal. Of course, the seeds may have undergone a number of preparation steps, including but not limited to cleaning, sorting, removing the shell (also referred to as "shelling") and heat treatment.

[0024] In a preferred embodiment, the method according to the invention is characterized in that the vegetable protein-rich material of step 1 is an isolate or a concentrate, preferably an isolate having a protein content greater than 70% by dry weight relative to the dry matter of the isolate, preferably between 75% and 95% by dry weight relative to the dry matter of the isolate. The protein content value may be 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 between each of these values. Advantageously, the vegetable protein-rich material of step 1 is a vegetable protein isolate or concentrate, preferably a vegetable protein isolate having a protein content greater than 80% by dry weight relative to the dry weight of the isolate, preferably between 80% and 90% by dry weight.

[0025] In a preferred embodiment, the method according to the invention is characterized in that the vegetable protein-rich material of step 1 originates from a legume, preferentially from a legume selected from the group consisting of peas and beans, more preferentially from peas.

[0026] In a preferred embodiment, the process according to the invention is characterized in that, before carrying out step 2, the pH of the suspension of vegetable protein-rich material of step 1 is modified so as to be between 5.0 and 7.5, preferentially between 6.5 and 7.5; preferentially so as to be equal to 7.0.

[0027] In a preferred embodiment, the process according to the invention is characterized in that the sugar content of the suspension of vegetable protein-rich material of step 1 is modified so as to contain between 1% and 10% sugars, preferentially between 2% and 8% sugars, even more preferentially between 3% and 6% sugars.

[0028] In a preferred embodiment, the method according to the invention is characterized in that the one or more strains of step 2 are selected from strains of Lactobacillus mucilaginosus, Lactobacillus mucilaginosus, Lactobacillus rhamnosus or Saccharomyces cerevisiae, preferentially strains of Lactobacillus mucilaginosus, Lactobacillus mucilaginosus or Lactobacillus rhamnosus. As explained below, due to taxonomic changes, the terms Lactobacillus mucilaginosus and Lactobacillus casei are interchangeable with the term Lactobacillus (see Zheng et al., "A taxonomic 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). Therefore, the strain of step 2 can be preferably selected from Lactobacillus mucilaginosus, Lactobacillus mucilaginosus or Lactobacillus rhamnosus.

[0029] In a preferred embodiment, the strain used in step 2 of the method of the invention has been cultured according to a method comprising:

[0030] i. inoculating a solid medium, such as agar, with a sample of the strain;

[0031] ii. pre-culturing the strain by incubating the inoculated solid medium of step 1 in an oven without stirring, preferably for between 10 and 24 hours;

[0032] iii. multiplying the strain by inoculating a liquid culture medium with a sample of the strain from the pre-culture of step ii and culturing the strain in the liquid culture medium.

[0033] In one embodiment, the incubation time of step iii is between 10 and 20 hours.

[0034] In a preferred embodiment, the method according to the invention is characterized in that step 3 is followed by step 4, which comprises the following sub-steps:

[0035] 4a. Optionally, modifying the pH of the suspension obtained at the end of step 3 to a pH between 5 and 9, preferentially to a pH between 6 and 9, preferentially to a pH of 7, preferentially by adding soda ash or lime to said suspension;

[0036] 4b. Heat-treating the suspension of step 3 or step 4a between 100°C and 160°C for 0.1 seconds to

[0037] 1 second, and

[0038] 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 the isolate.

[0039] The present invention also relates to a plant protein isolate obtainable by the method of the present invention.

[0040] In one embodiment, the vegetable protein isolate obtainable by the process of the present invention is characterized by a degree of hydrolysis (or DH) of less than 10%, preferentially less than 5%.

[0041] In one embodiment, the plant protein isolate obtainable by the method of the present invention is characterized in that the protein content of the plant protein isolate 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 weight of the dry matter of the isolate. The protein content value may be 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%. And any range between two of each of these values. Preferably, the plant protein isolate obtainable by the process of the present invention is characterized in that the protein content of the plant protein isolate is between 80% and 95% by weight, preferentially between 82% and 92% by weight, preferentially between 84% and 90% by weight, preferentially between 84% and 88% by weight, relative to the total weight of the dry matter of the isolate.

[0042] The present invention also relates to the use of the isolate obtainable by or obtained according to the process of the present invention for industrial applications including food, nutraceutical and pharmaceutical applications.

[0043] The present invention will be better understood by reading the following detailed description. DETAILED DESCRIPTION

[0044] The present invention relates to a method for improving the organoleptic properties of a material rich in vegetable protein, the method comprising the following steps:

[0045] 1. suspending a material rich in vegetable protein 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%;

[0046] 2. Add one or more microbial strains to the suspension of step 1 in order to obtain a suspension having the 5 cfu / mL and 1.10 10 cfu / mL, preferably 1.10 7 cfu / mL and 1.10 9 cfu / mL, preferably between 5.10 7 cfu / mL and 5.10 8 cfu / mL, the microbial strain is selected from a lactic acid bacteria strain, a yeast strain or a mixture thereof, the strain is preferably one or more lactic acid bacteria strains;

[0047] 3. Incubate the suspension of step 2 at a temperature between 20°C and 40°C for a duration between 30 and 120 minutes, preferentially between 45 and 75 minutes, even more preferentially between 50 and 70 minutes.

[0048] "Organoleptic" is understood to mean all stimuli related to the sensory domain, including taste, flavor, and smell. The sensory characteristics of compounds and / or foods can be evaluated using a sensory panel. A sensory panel consists of a group of people who will taste the compound and / or food according to a well-defined protocol. Each panelist will then give their sensory evaluation, which is typically composed of a descriptor (e.g., bitter, sweet, pea-like, vegetal, cardboard) and a score indicating the perceived intensity of the descriptor.

[0049] "Improving the organoleptic properties" or "improving the organoleptic quality" is understood in the present invention to mean a reduction in undesirable stimuli associated with plant protein-rich materials (e.g. associated with plant protein isolates), such as a vegetal flavor, typically an herbal flavor or a pea flavor ("beany" flavor). In the context of the present invention, a reduction in such undesirable stimuli can be achieved by converting compounds present in the plant protein-rich material, such as aldehydes, ketones and thiols, into other compounds (alcohols and / or carboxylic acids) under the action of one or more microbial strains described in the present application. In fact, these aldehydes, ketones and thiols are the main contributors to these undesirable stimuli, and the products of their biotransformation have, to a great extent, a flavor or odor that is less specific to plant protein-rich materials and / or a higher consumer perception threshold. For example, reducing the amount of green-note aldehydes leads to an organoleptic improvement of the product by reducing the "vegetal" sensory attributes.

[0050] In one embodiment, the method of the present invention for improving the sensory properties of plant proteins is a method for converting ketones and / or thiol aldehydes present in a plant protein-rich material into other compounds having a flavor or odor that is less characteristic of plant proteins and / or a higher perception threshold.

[0051] In one embodiment, the method of the present invention for improving the sensory properties of plant proteins is a method for converting some or all of the aldehydes with green notes present in the plant proteins into other compounds.

[0052] "Plant protein" is understood to mean any composition extracted from a natural raw material such as a seed, and preferably has been modified to make it more usable and more efficient for its formulation for final consumption. The modification may be very limited (e.g., simple grinding of the seed) or very complex (e.g., wet isolate manufacturing methods).

[0053] "Material enriched with vegetable protein" is understood to mean any powder, solution or flocculent material containing at least 1% by weight, preferably 25% by weight, of protein relative to the total weight of the material enriched with vegetable protein. Flours, concentrates, isolates, seeds may be mentioned in a non-limiting manner.

[0054] "Lactic acid bacteria" should be understood as meaning all Gram-positive, anaerobic bacteria that are partially oxygen-tolerant, generally do not form spores, take the form of shells or rods, and are capable of fermenting sugars to lactic acid. According to current taxonomic classification, they belong to the phylum Firmicutes, the class Bacilli, and the order Lactobacilli, which includes the following families: Aerococcaceae, Carnobacteriaceae, Enterococcus, Lactobacillaceae, Leuconostocaceae, and Streptococcaceae. The family Bifidobacteriaceae of the order Bifidobacteriaceae is also classified as lactic acid bacteria. Within the family Lactobacillaceae, the genus Lactobacillus is particularly important. Due to taxonomic changes, the terms Lactobacillus mucilaginosus and Lactobacillus casei are interchangeable with the term Lactobacillus (see Zheng et al., "A taxonomic 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 Lactobacillus mucilaginosus; Lactobacillus rhamnosus is now equivalent to Lactobacillus casei rhamnosus; and Lactobacillus reuteri is now equivalent to Lactobacillus mucilaginosus).

[0055] "Yeast" is understood to mean all unicellular fungi of variable shape (spherical, ovoid or ellipsoidal, flask-shaped, triangular or pointed (with each end swollen like a lemon), depending on the species, but generally ellipsoidal, about 6 to 10 microns and up to 50 microns, which reproduce by budding or fragmentation (schizogony). Most are related to the Ascomycetes (truffles, disc-type), some are related to another large group of higher fungi, the Basidiomycetes (Amanita, Boletus-type), and finally others are imperfect forms that cannot be clearly assigned to a definite group. They are usually able to complete sporulation, for the purpose of dormancy in adverse environments, or for the purpose of propagation. The common term "yeast" generally refers to the genus Saccharomyces (brewer's yeast or baker's yeast). There are many other types of yeast, the best known of which are Candida and Pichia.

[0056] "CFU" stands for "colony forming unit" and is used to estimate the number of living bacterial or fungal cells in a sample. Viability is defined as the ability to reproduce by cell division under controlled conditions.

[0057] "Cell density" is understood to mean the amount of bacterial or fungal cells present in a sample. It can be expressed in cfu / mL.

[0058] The suspension step 1 involves mixing the vegetable protein-rich material in a preferably aqueous solvent to obtain a vegetable protein suspension reaching between 5% and 20% dry matter, preferably between 7.5% and 15% dry matter, even more preferably between 8% and 12% dry matter. Thus, this value may be 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24% or 25%. And any range between each of these values.

[0059] In the present invention, the expression "protein content" or "protein abundance", for the most part relating to the protein-enriched material or composition according to the invention, designates the amount of nitrogen usually measured by the Kjeldhal method, multiplied by 6.25 and expressed as % by weight relative to the total weight of the protein-enriched material or composition according to the invention.

[0060] In a preferred embodiment, the process according to the invention is characterized in that the vegetable protein-rich material of step 1 is in the form of a vegetable powder (often called powder) having a protein content greater than 1% by weight, preferably between 10% and 40% by weight, preferably between 20% and 30% by weight, relative to the weight of the powder.

[0061] In this embodiment, the powder preferably consists essentially of seeds that have been ground and reduced to a powder. Of course, the seeds may have undergone a number of preparation steps including, but not limited to, cleaning, sorting, removing the hull (also known as "dehulling"), and heat treatment.

[0062] "Isolate" is understood to mean a protein-rich material having a protein content greater than 70%, preferentially greater than 80%, even more preferentially between 80 and 90% by dry weight relative to the total dry matter of the isolate.

[0063] “Concentrate” is understood to mean a protein-rich material having a protein content of between 25 and 70% by dry weight, preferentially between 35 and 60% by dry weight, even more preferentially between 40 and 50% by dry weight, relative to the total dry matter of the concentrate.

[0064] In a preferred embodiment, the method according to the invention is characterized in that the vegetable protein-rich material of step 1 is an isolate or a concentrate, preferably an isolate having a protein content greater than 70% by dry weight relative to the dry matter of the isolate, preferably between 75% and 95% by dry weight relative to the dry matter of the isolate. The protein content value may be 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 between each of these values.

[0065] In a preferred embodiment, the method according to the invention is characterized in that the vegetable protein-rich material of step 1 originates from a legume, preferentially from a legume selected from the group consisting of peas and beans, more preferentially from peas.

[0066] "Leguminosae" is understood to mean the dicotyledonous plant family of the order Fabaceae. This is one of the largest flowering plant families, second only to the Orchidaceae and Asteraceae in terms of species number. It contains approximately 765 genera, bringing together over 19,500 species. Several legumes are important crop plants, including soybeans, beans, peas, beans, chickpeas, peanuts, cultivated lentils, cultivated alfalfa, various clovers, broad beans, carob, and liquorice.

[0067] "Pea" is to be understood in its broadest sense and specifically includes all varieties of "smooth peas" and "wrinkled peas" and all mutant varieties of "smooth peas" and "wrinkled peas", regardless of the use (human food, animal feed and / or other uses) for which the variety is generally intended.

[0068] The term "pea" as used herein includes pea varieties belonging to the genus Pisum, more particularly Pisum sativum and Pisum aestivum. The mutant varieties are in particular those designated "mutant r", "mutant rb", "mutant rug 3", "mutant rug 4", "mutant rug 5" and "mutant lam", as described by C.L. HEYDLEY et al. in "Developing novel pea starches", Proceedings of the Symposium of the Industrial Biochemistry and Biotechnology Group of the Biochemical Society, 1996, pp. 77-87.

[0069] "Faba bean" is understood to be a family of annual plants of the species Vicia faba, belonging to the legume family of the Fabaceae, Fabaceae, Fabaceae, and Viciae. A distinction is made between minor and major varieties. In the present invention, both wild varieties and those obtained by genetic engineering or cultivar selection are excellent sources.

[0070] "Preferentially aqueous solvent" is understood to mean any solvent whose composition is predominantly water. Predominantly means that more than 50% by weight, preferably more than 90% by weight, even more preferably more than 95% by weight of the aqueous solvent is water, based on the weight of the solvent, or even that the aqueous solvent is "pure" water, i.e. a solvent consisting of 100% by weight of water.

[0071] "Water" should be understood to mean any so-called drinking water suitable for this purpose. "Drinking water" is understood to mean water that can be drunk or used for domestic and industrial purposes without health risks. Preferably, its conductivity is selected between 400 μS / cm and 1,100 μS / cm, preferably between 400 μS / cm and 600 μS / cm. More preferably, in the present invention, it should be understood that the drinking 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 in the range of between 6.5 and 9, and a total hardness (TH, i.e., the hardness of the water, corresponding to the measured value of the calcium and magnesium ion content in the water) of greater than 15 French degrees. In other words, drinking water must not have less than 60 mg / l of calcium or 36 mg / l of magnesium. This definition includes water from the drinking water network, decarbonated water, and demineralized water.

[0072] Thus, a suspension is obtained by intimately mixing the vegetable protein-rich material and an aqueous solvent. There are several methods of carrying out this process, including introducing a powder of the vegetable protein-rich material into the aqueous solvent (dry grinding followed by suspension), or introducing the vegetable protein-rich material into the aqueous solvent followed by a grinding step (suspension followed by wet grinding).

[0073] This mixture is prepared so as to obtain a vegetable protein suspension having a dry matter content (DM value) of between 5% and 20%, preferably between 7.5% and 15%, and even more preferably between 8% and 12%. The dry matter content is measured by any method known to the skilled person. It describes the percentage of solids in a mixture of substances. The higher the dry matter content, the drier the mixture.

[0074] Preferably, the so-called "drying" method is used: this method involves determining the amount of evaporated water by heating a known amount of a sample of known mass. A pre-weighed sample (called fresh material) is heated. Heating is continued until the mass stabilizes, indicating complete evaporation of the water. Preferably, a temperature of 105°C is used to allow any residual water to evaporate. The dried residue is weighed and expressed as a ratio. This gives the dry matter content of the sample analyzed.

[0075] Step 2 consists of adding one or more microbial strains to the suspension of the vegetable protein-rich raw material of step 1, the one or more strains being selected from the group consisting of lactic acid bacteria or yeast, preferably lactic acid bacteria, and the cell density of the suspension after the introduction of the one or more strains being between 1.10 and 2.0. 5 cfu / mL and 1.10 10 cfu / mL, preferably 1.10 7 cfu / mL and 1.10 9 cfu / mL, preferably between 5.10 7 cfu / mL and 5.10 8 cfu / mL; therefore, the cell density can 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 6cfu / mL、4.10 6 cfu / mL、5.10 6 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 10 cfu / mL、7.10 10 cfu / mL、8.10 10 cfu / mL、9.1010 cfu / mL and any ranges between these values ​​inclusive.

[0076] In a first embodiment, a single microbial strain is introduced into the suspension of the vegetable protein-rich feedstock of step 1 .

[0077] In a second embodiment, two or more microbial strains are introduced simultaneously into the suspension of the vegetable protein-rich feedstock of step 1 .

[0078] In a third embodiment, two or more microbial strains are introduced successively into the suspension of the vegetable protein-rich feedstock of step 1 .

[0079] To this end, in a first, so-called cultivation sub-step, the lactic acid bacteria or yeast are cultivated to achieve a cell density higher than that disclosed in the present application. A skilled person will know how to adapt the cultivation conditions to the selected strain in order to achieve this density. The cultivation step itself is subdivided into two steps: a first pre-cultivation step, the purpose of which is to revive the stored strain (e.g., frozen or lyophilized); and a second propagation step, the purpose of which is to propagate the strain to achieve the desired cell density.

[0080] According to one embodiment, the one or more strains used in step 2 have been cultured according to a method comprising:

[0081] i. inoculating a solid medium, such as agar, with a strain sample, preferably a frozen or lyophilized strain;

[0082] ii. pre-culturing the strain by incubating the inoculated solid medium of step 1 in an oven without stirring, preferably for 15 to 24 hours;

[0083] iii. multiplying the strain by inoculating a liquid culture medium with a sample of the pre-cultured strain of step ii and culturing the strain in the liquid culture medium.

[0084] Pre-cultivation and propagation are preferably carried out at a temperature suitable for the strain: typically at a temperature of about 35°C-38°C, preferably 38°C for lactic acid bacteria, and typically about 28°C-32°C for yeasts. Advantageously, the incubation time of propagation step iii is relatively long, typically between 12 and 20 hours, preferably about 15 hours; and the propagation time is between 8 and 15 hours, and preferably at least about 10 hours. In fact, as shown in the experimental section, a minimum propagation time of 10 hours is preferred to achieve the maximum technical effect of the present invention.

[0085] The preferred culture regimen is as follows:

[0086] i. Inoculation step: Take out the frozen strain sample from the cryovial and inoculate it into the appropriate culture medium

[0087] (usually agar), which is contained in a tube, for example a 10 mL tube;

[0088] ii. Pre-incubation step: Incubate the tube in an oven without stirring for 15 hours to

[0089] 24 hours.

[0090] iii. Propagation step: The pre-culture tubes are then used to inoculate containers (flasks or Erlenmeyer flasks) at 1 vol / vol %, containing for example 1 L of the culture medium used in the above examples in 10 mL tubes.

[0091] The cultivation was carried out at the optimal temperature for each strain, for the optimal time for each strain, as detailed in Table 1 for the lactic acid bacteria and yeast examples.

[0092] The second sub-step then consists of introducing a determined amount of the culture into another amount of the suspension enriched with vegetable protein prepared in step 1, so as to finally obtain a suspension of 1.10 5 cfu / mL and 1.10 10 cfu / mL, preferably 1.10 7 cfu / mL and 1.10 9 cfu / mL, preferably between 5.10 7 cfu / mL and 5.10 8 cfu / mL.

[0093] For example, will have 1.10 9 cfu / mL titer of 10mL lactic acid bacteria culture was introduced into 90mL of plant protein-rich suspension, which would produce a 8 100 mL suspension of a cell density of cfu / mL

[0094] In a preferred embodiment, the method according to the present invention is characterized in that the one or more strains of step 2 are selected from fermentative mucilaginous lactobacillus, mucilaginous lactobacillus reuteri, rhamnosus lactobacillus or saccharomyces cerevisiae, preferentially fermentative mucilaginous lactobacillus, mucilaginous lactobacillus reuteri or rhamnosus lactobacillus. Due to taxonomic changes, the terms mucilaginous lactobacillus and lactobacillus also refer to lactobacillus (see the article by 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 disclosed above, can be introduced into the suspension together. Therefore, the strain of step 2 can be preferentially selected from fermentative mucilaginous lactobacillus, mucilaginous lactobacillus reuteri or rhamnosus lactobacillus.

[0095] In a first embodiment, a single microbial strain selected from Lactobacillus mucilaginosus, Lactobacillus mucilaginosus, Lactobacillus rhamnosus or Saccharomyces cerevisiae, preferentially Lactobacillus mucilaginosus, Lactobacillus mucilaginosus or Lactobacillus rhamnosus, is introduced into the suspension of the vegetable protein-rich raw material of step 1.

[0096] In a second embodiment, two or more microbial strains selected from Lactobacillus mucilaginosus, Lactobacillus mucilaginosus, Lactobacillus rhamnosus or Saccharomyces cerevisiae, preferentially Lactobacillus mucilaginosus, Lactobacillus mucilaginosus or Lactobacillus rhamnosus, are introduced simultaneously into the suspension of the vegetable protein-rich raw material of step 1.

[0097] Therefore, the combination of strains can be

[0098] - Lactobacillus mucilaginosus and Lactobacillus mucilaginosus ferments,

[0099] - Lactobacillus mucilaginosus and Lactobacillus rhamnosus ferments,

[0100] - Fermentation of Lactobacillus mucilaginosus and Saccharomyces cerevisiae,

[0101] - Lactobacillus reuteri and Lactobacillus rhamnosus,

[0102] - Lactobacillus reuteri and Saccharomyces cerevisiae,

[0103] - Lactobacillus rhamnosus and Saccharomyces cerevisiae,

[0104] - Lactobacillus mucilaginosus fermented with Lactobacillus mucilaginosus and Lactobacillus rhamnosus,

[0105] - Lactobacillus mucilaginosus, Lactobacillus mucilaginosus and Saccharomyces cerevisiae

[0106] - Fermented Lactobacillus mucilaginosus, Lactobacillus rhamnosus and Saccharomyces cerevisiae

[0107] - Lactobacillus reuteri, Lactobacillus rhamnosus and Saccharomyces cerevisiae

[0108] - Lactobacillus mucilaginosus, Lactobacillus mucilaginosus, Lactobacillus rhamnosus and Saccharomyces cerevisiae

[0109] In a third embodiment, two or more microbial strains selected from Lactobacillus mucilaginosus, Lactobacillus mucilaginosus, Lactobacillus rhamnosus or Saccharomyces cerevisiae, preferentially Lactobacillus mucilaginosus, Lactobacillus mucilaginosus or Lactobacillus rhamnosus, are continuously introduced into the suspension of the vegetable protein-rich raw material of step 1.

[0110] Therefore, the combination of strains can be

[0111] - Lactobacillus mucilaginosus, then Lactobacillus mucilaginosus, and vice versa

[0112] - Fermentation of Lactobacillus mucilaginosus, then Lactobacillus rhamnosus, and vice versa

[0113] - Fermentation of Lactobacillus mucilaginosus, then Saccharomyces cerevisiae, and vice versa

[0114] - Lactobacillus reuteri, then Lactobacillus rhamnosus, and vice versa

[0115] - Lactobacillus reuteri, then Saccharomyces cerevisiae, and vice versa

[0116] - Lactobacillus rhamnosus, then Saccharomyces cerevisiae, and vice versa

[0117] An example of such a strain from a deposited reference is Lactobacillus fermentum CIP 102980 T or CNCM I-5802 (now Lactobacillus mucilaginosus); Lactobacillus rhamnosus LMG 18243 or ATCC 53103, also known as Lactobacillus rhamnosus GG (now Lactobacillus rhamnosus); Lactobacillus reuteri DSM 17509 (now Lactobacillus mucilaginosus); Saccharomyces cerevisiae CBS 8066.

[0118] Preferred culture parameters are described in Table 1:

[0119] Table 1

[0120]

[0121] (MRS = De Man, Rogosa and Sharpe agar; MW = Malt Wickerham agar)

[0122] As will be illustrated in the Examples section of the description below, propagation of less than 10 hours or more than 20 hours will not achieve the maximum expected effect. Surprisingly, the proprietors have found that this incubation time is necessary for the strain to be active.

[0123] Preferably, at the end of the first incubation period and before the second step of introducing a certain amount of the strain into the plant protein suspension, the culture is washed to remove excess culture medium. The culture is centrifuged at 4000 g and 4°C for 10 minutes. The supernatant is removed and the pellet is resuspended in water. A new centrifugation is then performed and the supernatant removed. The strain is then resuspended in a volume of water based on the calculated biomass, so that it can be grown at a constant volume within 10 minutes. 6 CFU / mL and 10 9 CFU / mL, preferably between 10 7 CFU / mL and 10 8 CFU / mL, or even more preferably 10 8 CFU / mL (i.e., if the suspension is inoculated at 10%, then 10 9 CFU / mL, aiming to obtain 10 8 The test medium was inoculated with a final cell density of 100 CFU / mL.

[0124] Contacting step 3 consists of incubating the inoculated suspension of step 2 at 30°C + / - 10°C for between 30 and 120 minutes, preferentially between 45 and 75 minutes, even more preferentially between 50 and 70 minutes. The temperature 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, and any range between each of these values. The contact time can be 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, and any range between each of these values.

[0125] Surprisingly and unexpectedly, the applicants have found that contact times between 30 and 120 minutes are sufficient to reduce undesirable flavors and also produce new desirable flavors while limiting modifications of the protein, such as enzymatic hydrolysis.

[0126] A skilled person would never consider a short time to achieve these conversions, since this time corresponds to the incubation period required for the strain to adapt before catabolism is restored. Very low amounts of sugar also appear to be too low for effective catabolism recovery. Since volatile compounds are extracellular, they must come into contact with intracellular enzymes, be modified, and then be released. This transport mechanism has not yet been clearly identified, but its complexity seems incompatible with the short bioconversion times according to the present invention.

[0127] The incubation may be static or agitated, in the latter case the agitation must be sufficient to keep the suspension homogeneous but not too high as this may degrade the cells.

[0128] In a preferred embodiment, the process according to the invention is characterized in that, before carrying out step 2, the pH of the suspension of the vegetable protein-rich material of step 1 is modified so as to be between 5.0 and 7.5, preferentially between 6.5 and 7.5; preferentially so as to be equal to 7.0. Thus, the pH may 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. And any range between each of these values.

[0129] In a preferred embodiment, the method according to the invention is characterized in that the suspension of the vegetable protein-rich material of step 1 contains between 0.1% and 1% total sugars, preferably between 0.2% and 0.8% total sugars, even more preferably between 0.3% and 0.6% total sugars. The total sugar value 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 between each of these values.

[0130] "Sugar" is understood to mean a group of carbohydrates selected from monosaccharides such as glucose, fructose, ribose, galactose and mixtures thereof and oligosaccharides such as raffinose, sucrose, melezitose, stachyose and mixtures thereof.

[0131] For the purposes of the present invention, "monosaccharide" is understood to mean a carbohydrate consisting of a single molecule, such as glucose, fructose, mannose, galactose, xylose and arabinose.

[0132] For the purposes of the present invention, the term "oligosaccharide" means an oligomer formed by a number n of sugars (monosaccharides) via α or β glycosidic bonds. By convention, the number n varies between 3 and 10. In this context, they are located between single sugars (n=1) and polysaccharides (polysaccharides) (n>10). However, this limit of 10 units is not completely fixed, and polysaccharides with a degree of polymerization of 11 to 25 are generally assimilated to them. Oligosaccharides comprising 2 sugars are disaccharides (sucrose), those comprising 3 sugars are trisaccharides (raffinose, melezitose), and those comprising 4 sugars are tetrasaccharides (stachyose). Oligosaccharides can be linear (stachyose), branched or cyclic (cyclodextrins).

[0133] For the purposes of the present invention, "total sugars" is understood to mean the monosaccharide composition obtained after complete hydrolysis of sugars. Oligosaccharides and polysaccharides are completely hydrolyzed chemically (acid or base) and / or enzymatically (amylase), and the hydrolyzed samples are then analyzed to determine the composition of the monosaccharides obtained.

[0134] In a preferred embodiment, the total sugars include a mixture of glucose, fructose, mannose, galactose, xylose, and arabinose. Preferably, the total sugars include between 20% and 30% glucose, between 20% and 30% galactose, and between 20% and 30% arabinose. For example, a typical total sugar composition suitable for use in the present 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.

[0135] The total sugars present in the suspension of the plant protein-rich material of step 1 are partly derived 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%.

[0136] It should be noted that not all isolates contain these sugars, in particular mixtures of sucrose, raffinose, melezitose and stachyose. Application EP 3 071 046 B1 proposes, for example, an extraction process comprising a seed soaking step which virtually eliminates sugars including pea GOS (galacto-oligosaccharides).

[0137] Then, step 4 comprising sub-steps may also be performed. The purpose of these sub-steps is to isolate the vegetable protein, extract it and stabilize it for its subsequent use.

[0138] Thus, in one embodiment, step 3 of the method according to the invention is followed by step 4 comprising the following sub-steps:

[0139] 4a. Optionally, modifying the pH of the suspension obtained at the end of step 3 to a pH between 5 and 9, preferentially to a pH between 6 and 9, preferentially to a pH of 7, preferentially by adding soda ash or lime to said suspension;

[0140] 4b. Heat-treating the suspension of step 3 or step 4a between 100°C and 160°C for 0.1 seconds to

[0141] 1 second, and

[0142] 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 the isolate.

[0143] Step 4b generally corresponds to HTST or UHT heat treatment.

[0144] Starting from a powder or a powdered material, step 4 may, for example, comprise the following sub-steps:

[0145] 4a. Removal of the insoluble fraction (by internal centrifugation, mainly containing starch and fiber),

[0146] 4b. Isoelectric precipitation of the globulin fraction and its recovery by centrifugation,

[0147] 4c. pH neutralized to 7,

[0148] 4d.HTST or UHT heat treatment;

[0149] 4e. Dry using atomizer.

[0150] In a preferred embodiment, starting from the isolate, the method according to the invention is characterized in that step 3 is followed by the following steps:

[0151] 4a. Optionally, modifying the pH of the suspension obtained at the end of step 3 to a pH between 6 and 9, preferentially to a pH of 7, preferentially by adding soda ash or lime to said suspension;

[0152] 4b. Optionally, heat treating the suspension of step a to between 100°C and 160°C for 0.1 to 1 second, and

[0153] 4c. Drying the plant powder suspension of step b by means of an atomizer until a plant powder isolate having a dry matter content of greater than 95% is obtained.

[0154] The present invention also relates to a vegetable protein isolate obtainable by the process of the present invention.

[0155] The present invention also relates to a vegetable protein isolate obtainable by the process of the invention characterized in that its degree of hydrolysis (or DH) is less than 10%, preferentially less than 5.

[0156] The term "isolate obtainable according to the method of the invention" is used interchangeably with "isolate according to the invention".

[0157] Isolates according to the present invention have a degree of hydrolysis (or DH) of less than 10%, preferably less than 5%, including DHs of 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% and 1%, and any ranges therebetween, inclusive.

[0158] The degree of hydrolysis can be determined by measuring the free amino nitrogen content by the OPA method disclosed in the article by Nielsen et al. 2001 (Nielsen et al., Journal of Food Science, Vol. 66, Issue, pp. 642-646, 2001 "Improved Method for Determining Food Protein Degree of Hydrolysis") relative to the total nitrogen measured by the DUMAS method according to standard ISO 16634-2:2016.

[0159] Particular preference is given to the following test for measuring the degree of hydrolysis. Its principle consists first of determining the amino nitrogen content (free NH2 functional groups) of the protein sample (preferably using the MEGAZYME kit (reference K-PANOPA)), then determining the protein nitrogen content (total nitrogen, therefore including free and bound NH2 functional groups) of the sample, and finally calculating the degree of hydrolysis, which is obtained by the ratio of these two measurements.

[0160] Determination of amino nitrogen content :

[0161] The amino nitrogen groups of free amino acids in the sample react with N-acetyl-L-cysteine ​​and o-phthalaldehyde (OPA) to form isoindole derivatives.

[0162] The amount of isoindole formed during this reaction is stoichiometric to the amount of free amino nitrogen. It is an isoindole derivative, measured by the increase in absorbance at 340 nm.

[0163] Place an accurately weighed sample P* of the sample to be analyzed in a 100 ml beaker. Based on the amino nitrogen content of the sample, the sample will be 0.5 g to 5.0 g. Add approximately 50 ml of distilled water, homogenize and pour the mixture into a 100 ml graduated flask. Add 5 ml of 20% sodium dodecyl sulfate (SDS) and make up the mixture to a volume of 100 ml with distilled water. Stir with a magnetic stirrer at 1000 rpm for 15 minutes. Solution No. 1 is prepared by dissolving the tablet from bottle 1 of the Megazyme test kit in 3 ml of distilled water and stirring until it is completely dissolved. One tablet is required for each test. Solution No. 1 is prepared extemporaneously before use.

[0164] Prepare blanks, standards, and samples directly in the spectrophotometer cuvettes under the following conditions:

[0165] -Blank: Add 3.00ml of solution 1 and 50μl of distilled water

[0166] - Standard: Add 3.00 ml of solution 1 and 50 μl of bottle 3 of the Megazyme kit

[0167] - Sample: Add 3.00 ml of Solution 1 and 50 μl of Sample Preparation Solution.

[0168] The contents of each cuvette were mixed and absorbance measurements (A1) of the solutions were taken approximately 2 min after placement in a spectrophotometer at 340 nm (the spectrophotometer was equipped with a cuvette with a 1.0 cm pathlength, capable of measuring at a wavelength of 340 nm, and validated according to the procedures described in the relevant manufacturer's technical manual).

[0169] The reaction was then immediately initiated by adding 100 μl of solution No. 2 (corresponding to the OPA solution from bottle 2 of the Megazyme kit) to each spectrophotometer cuvette.

[0170] The contents of each cuvette were mixed and then placed in the dark for approximately 20 minutes.

[0171] Then obtain absorbance measurements A2 of the blank, standards, and samples from the spectrophotometer at 340 nm.

[0172] The free amino nitrogen content, expressed as a weight percentage relative to the weight of the product, is given by the formula:

[0173] Formula 1

[0174]

[0175] Formula 2

[0176]

[0177] in:

[0178] ΔAech=Aech2–Aech1

[0179] ΔAblc=Ablc2–Ablc1

[0180] Aech2 = absorbance of the sample after adding solution 2

[0181] Aech1 = absorbance of the sample after adding solution 1

[0182] Ablc2 = absorbance of blank after adding solution 2

[0183] Ablc1=Absorbance of blank after adding solution 1

[0184] V = volume of the flask

[0185] m = weight of the sample (in g)

[0186] 6803 = Extinction coefficient of isoindole derivatives at 340 nm (in 1.mol -1 .cm -1 as units).

[0187] 14.01 = molar mass of nitrogen (in g.mol -1 (unit)

[0188] 3.15 = Final volume in the cuvette (in ml)

[0189] 0.05 = Sample in the cuvette (in ml)

[0190] Determination of protein nitrogen content :

[0191] The protein nitrogen content is determined according to the DUMAS method in accordance with ISO 16634:2016. It is expressed as a weight percentage relative to the weight of the product.

[0192] Calculation of degree of hydrolysis :

[0193] The degree of hydrolysis (DH) was calculated using the following formula:

[0194] Formula 3

[0195]

[0196] The isolate obtainable according to the process of the invention may also be characterized by a reduced content of volatile compounds causing undesirable irritation.

[0197] In one embodiment, the isolate obtainable according to the process of the invention is characterized by a reduced content of volatile compounds responsible for vegetal flavours, such as herbal flavours or pea flavours (“beany” flavours).

[0198] The organic compound responsible for the plant flavor can be a compound comprising one or more aldehyde groups and / or ketone groups. In the present application, the term "aldehyde" can be used to refer to a compound comprising one or more -CHO groups, preferably 1 or 2 -CHO groups, and even more preferably only 1 -CHO group. The term "ketone" can be used to refer to a compound comprising one or more -CO-R groups, preferably one or two -CO-R groups, and even more preferably a single -CO-R group.

[0199] Examples of aldehydes responsible for vegetal flavors are leafy aldehydes such as hexanal, nonanal, 2-nonenal, benzaldehyde, and 3-methylbutanal.

[0200] An example of a ketone responsible for vegetal flavor is 3-octen-2-one.

[0201] The organic compound responsible for the vegetal flavor may also be an alcohol, such as 1-octen-3-ol, or a pyrazine, such as 2,5-dimethylpyrazine.

[0202] In one embodiment, the isolate obtainable according to the process of the invention has a reduced content of at least 1, 2, 3 or 4 volatile compounds responsible for the vegetal flavour, the volatile compounds being chosen from hexaldehyde, nonanal, 2-nonenal, benzaldehyde, 3-methylbutanal, 3-octen-2-one, 1-octen-3-ol and 2,5-dimethylpyrazine.

[0203] The presence of these compounds in conventional commercial isolates, even at low levels, can give them so-called "vegetal" flavors, such as pea flavor, which is undesirable when foods are prepared using the isolates. The process according to the invention preferably makes it possible to obtain an isolate having a content of at least volatile compounds responsible for the vegetal flavour reduced by more than 50%, preferentially more than 25%, this reduction 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 between each of these values.

[0204] In one embodiment, the isolate obtainable by the process of the invention is characterized by a reduced content of compounds comprising one or more thiol groups, in particular a reduced content of methyl mercaptan.

[0205] This compound, derived from the degradation of sulfur-containing amino acids, is synthesized in conventional commercial isolates. When the isolates are used to prepare ready-to-drink beverages or wet-extruded strips intended for meat analogs, the compound is present at ppb levels sufficient to produce flavors known as "rancid egg" or "hydrogen sulfide." Without wishing to be bound by a particular theory, these processes generate significant amounts of heat, leading to the presence of significant amounts of methyl mercaptan-derived degradation compounds. The process according to the invention preferably makes it possible to obtain an isolate whose methyl mercaptan content is reduced by more than 30%, 40%, 50%, preferentially by more than 50%, possibly by 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%.

[0206] In one embodiment, the isolate obtainable by the process of the invention is characterized by a reduced content of volatile compounds responsible for vegetal flavor and a reduced content of methyl mercaptan. In the present application, the term "thiol" may be used to denote a compound comprising at least one -SH group, preferably comprising 1 or 2 thiol groups, even more preferably comprising a single thiol group.

[0207] The plant protein isolate according to the invention is characterized in that its protein content (expressed relative 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%. Thus, the plant protein isolate according to the invention will be considered to have a protein content (expressed relative to its total dry matter) of 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% or 95%. And any range between each of these values ​​included.

[0208] The present invention also relates to the use of an isolate according to the invention or an isolate obtained according to a process according to the invention for industrial applications including food, nutraceutical and pharmaceutical applications.

[0209] The human and animal food industry is understood to mean industrial confectionery (e.g. chocolate, caramel, jelly candies), bakery products (e.g. bread, brioches, muffins), the meat and fish industry (e.g. sausages, burgers, fish nuggets, chicken nuggets), sauces (e.g. pâté, mayonnaise), products of milk origin (e.g. cheese, vegetable milks), beverages (e.g. high-protein drinks, powdered drinks to be reconstituted).

[0210] In general, the plant protein-rich material whose organoleptic properties have been improved by the method of the present invention, in other words the composition according to the present invention, in particular the isolate according to the present invention, can be used in foods in an amount of up to 100% by weight relative to the total dry weight of the food, for example in an amount of from about 1% to about 80% by weight relative to the total dry weight of the food or beverage. All intermediate amounts are envisaged (i.e. 2%, 3%, 4%, ... 77%, 78%, 79% by weight relative to the total weight of the food or beverage), as well as all intermediate ranges based on these amounts. Food products that may be contemplated in the context of the present invention include baked products; baked products (including but not limited to rolls, pastries, pies, cakes and cookies); pre-made sweet baking mixes for preparing sweet baked products; pie fillings and other sweet fillings (including but not limited to fillings for fruit pies and fillings for nut pies, such as fillings for pecan pie, and fillings for cookies, cakes, pastries, confectionery products and similar products, such as fillings for fat-based creams); desserts, gelatin and puddings; frozen desserts (including but not limited to frozen dairy desserts such as ice cream (including regular ice cream, soft ice cream and all other types of ice cream), and non-dairy frozen desserts (such as non-dairy ice cream, sorbet and similar products)); carbonated beverages (including but not limited to carbonated soft drinks); non-carbonated beverages (including but not limited to non-carbonated soft drinks, such as flavored beverages), juices and beverages based on sweetened tea or coffee); beverage concentrates (including but not limited to liquid concentrates and syrups and non-liquid concentrates, such as freeze-dried preparations and / or powdered preparations yogurt (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 of these products); snack bars (including but not limited to cereal bars, nut bars, grain bars and / or fruit bars); bread products (including but not limited to leavened and unleavened breads, breads with yeast and without yeast (such as soda bread), breads containing any type of wheat flour, breads composed of any type of non-wheat flour (such as potato, rice and rye flour), and breads that do not contain gluten); for preparing Pre-prepared bread mixes for bakery products; sauces, syrups and vinaigrettes; sweetened spreads (including but not limited to pectin, jams, butters, nut spreads and other spreadable preserves, candied fruits and other similar products); confectionery products (including but not limited to jelly beans, soft candies, hard candies, chocolates and chewing gum); sugar-coated and sugar-free coated breakfast cereals (including but not limited to extruded breakfast cereals, flaked breakfast cereals and puffed breakfast cereals) and cereal coating compositions intended for use in preparing sweetened breakfast cereals. Other types of food and beverage products not mentioned herein but which typically contain one or more nutritive sweeteners are also contemplated in the context of the present invention. In particular, animal feed (such as pet food) is explicitly contemplated.It can also be used in meat products such as emulsified sausages or veggie burgers after texturization by extrusion.It can also be used in egg replacer formulations.

[0211] The composition of the present invention can be used as a single protein source, but can also be used in combination with other plant or animal proteins. The term "plant protein" means all proteins derived from cereals, oil plants, leguminous plants and tuberous plants, as well as all proteins derived from algae and microalgae or fungi, which are used alone or as a mixture, selected from the same family or different families. In this application, the term "cereal" refers to plants cultivated from the grass family that produce edible grains, such as wheat, rye, barley, corn, sorghum or rice. Grains are often ground into powder form, but are also provided in the form of cereals, and are sometimes provided in the form of whole plants (feed crops). In this application, the term "tuber" encompasses storage organs that are typically located underground, which ensure the survival of plants in winter and the reproduction that they often undergo through nutritional processes. These organs are spherical due to the accumulation of stored material. The organ to be transformed into a tuber can be a root (e.g., carrot, parsnip, cassava, konjac), a rhizome (e.g., potato, Jerusalem artichoke, Japanese artichoke, sweet potato), the base of a stem (more particularly the hypocotyl, e.g., kohlrabi, celery root), a root and hypocotyl combination (e.g., beetroot, radish). For the purposes of the present invention, the term "leguminous plant" refers to any plant belonging to the Cesalpiniaceae, Mimosaceae or Papilionaceae families, and in particular: all plants belonging to the Papilionaceae family, such as peas, beans, soybeans, beans, mung beans, mung beans, lentils, alfalfa, clover or lupins. This definition specifically includes all plants described in a table from 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 proteins or milk proteins, such as whey proteins, caseins or caseinate proteins. Thus, pea protein compositions can 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 modified

[0212] The present invention relates largely to the use of the isolate according to the invention for the manufacture of biscuits, muffins, pancakes, nutritional bars (intended for specialized nutrition / weight loss or sports nutrition), in protein-enriched bread or gluten-free bread, in high-protein mini-cereals ("crispies") obtained by cooking-extrusion, wherein more particularly a high-protein solution is sought which has no negative impact on the preparation process or on the texture of the final preparation or product.

[0213] For the purposes of the present invention, "powdered nutritional preparations" are powdered preparations comprising at least, preferably exclusively, a leguminous plant protein according to the invention, and in particular pea or bean protein, which can be reconstituted with an aqueous liquid and are suitable for oral administration to humans.

[0214] Unless otherwise indicated, the expression "dry mix" as used herein refers to the mixing of components or ingredients to form a base nutritional powder, or to the addition of dry powdered or granular components or powder-based ingredients to form a powdered nutritional formulation.

[0215] All percentages, parts and ratios as recited herein are by weight of the total formulation, unless otherwise specified.

[0216] The powdered food formulations and corresponding manufacturing methods of the present invention may comprise, consist of, or consist essentially of the basic elements of the invention as described herein and any additional or optional elements described herein or otherwise useful in nutritional formulation applications.

[0217] The powdered nutritional formulations of the present invention are typically in the form of a flowable or substantially fluid granular composition, or at least a granular composition that can be easily molded and measured using a spoon or another similar device, wherein the composition can be easily 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, "immediate" use typically means within about 48 hours, more typically within about 24 hours, preferably directly after reconstitution.

[0218] Powdered food preparations may be formulated with all types and amounts of nutrients sufficient to form a dietary supplement, or a specialized nutritional preparation intended for people following specific diets designed for sports nutrition and weight loss.

[0219] In an exemplary embodiment, the powdered nutritional formulation may be formulated for:

[0220] - Repair muscles after intense effort, such as in athletes,

[0221] -Maintain or build muscle mass in athletes, or

[0222] - As a meal replacement for people trying to lose weight through its satiogenic effect.

[0223] Powdered food preparations may have a caloric density suitable for the nutritional needs of the end user, but in most cases the reconstituted powder will have from about 350 kcal to about 400 kcal per 100 ml.

[0224] Powdered food formulations can have a protein content suitable for the nutritional needs of the end user, but in most cases the reconstituted powder includes from about 20 g to about 91 g protein per 100 g, including from about 40 g to about 65 g protein per 100 g.

[0225] Thus, the formulation may comprise 20% to 95% protein, such as 20%-90%, 30%-80% or 40%-60% relative to the total weight of the formulation.

[0226] For example, the legume protein isolate (preferably pea or bean protein isolate) according to the present invention may comprise 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, or 90%-100% of the total protein content of the formulation, or any combination of these percentage ranges. 100% is the ultimate preferred embodiment for maximizing the effect of FGF19 overexpression.

[0227] Furthermore, the powdered food formulation may have a fat content suitable for the nutritional needs of the end user, but in most cases the reconstituted powder comprises from about 0.5 g to about 13 g per 100 g, including from about 3 g to about 7 g per 100 g. Thus, the formulation may comprise from 0 to 20% lipid, for example 0.5%-15%, 1%-10% or 3%-7% (particularly by weight), relative to the total weight of the formulation.

[0228] The powdered nutritional formulations of the present invention can be packaged and sealed in single or multiple use containers and then stored at ambient conditions for up to about 36 months or longer, more typically from about 12 months to about 24 months.

[0229] For multiple uses of the containers, they can be opened and closed for repeated use by the user, provided that the closed package is then stored under ambient conditions (e.g., avoiding extreme temperatures) and the contents are used within about one or two months.

[0230] In particular, the nutritional preparations according to the invention can be used in the following areas:

[0231] -Diet and nutrition (exercise, weight loss),

[0232] - clinical nutrition (in the form of a drink, dessert cream or enteral bag),

[0233] - Dairy products (in the form of yogurt, dairy drinks, dairy cream, frozen desserts or sorbets).

[0234] -Biscuit products, pastry products, bread-making products and high-protein cereal products.

[0235] In the athletic field, protein is known to play a role in muscle maintenance and growth. Protein intake is also important for athletes who practice weightlifting or resistance training.

[0236] These proteins must be balanced in terms of their amino acid profile and must comply with FAO / WHO recommendations. Their digestibility is an important factor, ranging from rapidly to slowly digestible, depending on the time of protein intake.

[0237] Ready-to-drink protein-enriched or high-protein beverages thus provide the body with an intake of selected proteins with fewer calories.

[0238] These high-protein drinks must:

[0239] -Rich in protein, low in carbohydrates and fat;

[0240] -Has a good taste;

[0241] - Designed to aid weight loss, stimulate fat burning and aid muscle recovery;

[0242] - is full;

[0243] -Can be used to manage cravings without any added sugar or fat;

[0244] -Has a balanced content of essential amino acids, fiber, vitamins and minerals;

[0245] -Low calories.

[0246] These ready-to-drink beverages can advantageously be prepared with the soy protein isolates according to the invention, preferably bean or pea isolates. In addition, they can be used as the sole protein source, preferably because they maximize the overexpression effect of FGF19.

[0247] For example, plant-based beverages that provide milk alternatives contain on average 4.5g to 11g of protein per 100ml of beverage, preferably around 7g of protein per 100ml, and are very low in fiber (around 0.5g to 1g per 100ml).

[0248] Thus, the beverage may comprise from 1% to 20% protein relative to the total weight of the beverage, such as 3%-15% or 6%-8%.

[0249] For example, the pea protein isolate according to the present invention can comprise 50%-60%, 60%-70%, 70%-80%, 80%-90%, or 90%-100% of the total protein, or any combination of these percentage ranges. Preferably, it comprises at least 52%. In particular, the pea protein intake is 52% to 100% of the total protein intake.

[0250] For ready-to-drink beverages, the pea protein intake can be in the range of 0 to 100%, preferably in the range of 0.01% or 0.1% to 100%. For example, the pea protein isolate according to the present invention can comprise 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.

[0251] In the field of "diet" drinks, i.e., those intended for use in calorie-restricted or weight-loss diets, as mentioned above, these high-protein drinks or protein-rich drinks are not only effective for rapid muscle growth. This type of drink is also very advantageous in the context of a weight-loss diet based on protein consumption.

[0252] Diet drinks are known to be ideal for aiding weight loss. More specifically, they make it possible to:

[0253] - Provides a satiety effect

[0254] - Protects muscles and tones the body to avoid weight gain.

[0255] Like "sports" drinks, these diet drinks have:

[0256] -Balanced content of essential amino acids, fiber, vitamins and minerals

[0257] - Low in sugar, fat and calories.

[0258] Thus, protein-rich drinks are actually very effective for quickly losing several kilograms. These protein-rich preparations simply reduce or prevent the hunger of the person consuming them. By drinking such drinks, for example, users can significantly reduce the amount of food they eat and can quickly lose weight (in the context of a meal replacement program for weight control, or as a substitute for the total daily food intake for weight control).

[0259] In clinical nutrition, enteral nutrition is known as a therapeutic solution fed through a tube, which is used when the digestive tract is functional and accessible but the patient cannot eat normally, or in cases of severe malnutrition.

[0260] This technology allows nutrients to be supplied directly into the digestive tract. It completely or partially replaces conventional oral feedings with "complete" nutritional formulas that provide all the nutrients the body needs.

[0261] These formulations are typically packaged in flexible bags (made of PVC) and administered through a nasogastric tube, gastrostomy, nasojejunal tube, nasoduodenal tube, or jejunostomy.

[0262] These nutrient blends consist of proteins, lipids, carbohydrates, vitamins, and minerals, with or without fiber.

[0263] A distinction is made between several categories: polymer mixtures (standard) and semi-elemental mixtures ("predigested"), the latter being indicated in very specific cases (short bowel syndrome, exocrine pancreatic insufficiency, etc.):

[0264] -Polymer mixture

[0265] - Low calorie (0.5kcal / mL-0.75kcal / mL), normal or high protein, with or without fiber

[0266] - Isocaloric (1 kcal / mL), normal or high protein, with or without fiber

[0267] - High calorie (1.25kcal / mL-1.5kcal / mL), normal or high protein, with or without fiber

[0268] -Specific formulations (disturbance of blood sugar metabolism, respiratory failure).

[0269] Semi-elemental blends are isocaloric or hypercaloric, normal or high protein blends based on medium chain peptides and triglycerides.

[0270] Pea protein isolate as a protein source is particularly suitable for this purpose due to its functional properties.

[0271] Furthermore, they make it possible to maintain the same properties as milk proteins at a lower cost.

[0272] The present invention will be better understood by the following examples, which are intended only to better understand the present invention. These examples are not intended to limit the scope. BRIEF DESCRIPTION OF THE DRAWINGS

[0273] Other features, details and advantages of the present invention will become apparent upon reading and analyzing the accompanying drawings, in which:

[0274] [ Figure 1 ] shows that in Effect of strain selection from Lactobacillus fermentans, Lactobacillus reuteri, Lactobacillus rhamnosus, and Lactobacillus rhamnosus (Lr) on their efficacy in modifying volatile compounds after F85F contact.

[0275] [ Figure 2 ] shows that in Effect of cultivation time of Lactobacillus fermentans (Lf) and Lactobacillus rhamnosus (Lr) strains before F85F exposure on their efficacy in improving sensory quality. F85F; B = white; Lf 5h = Lactobacillus fermentum precultured for 5 h; Lf 10h = Lactobacillus fermentum precultured for 10 h; Lf 15h = Lactobacillus fermentum precultured for 15 h; Lr 5h = Lactobacillus rhamnosus precultured for 5 h; Lr 10h = Lactobacillus rhamnosus precultured for 10 h; Lr 15h = Lactobacillus rhamnosus precultured between 5 h and 15 h.

[0276] [ Figure 3 ] show the effect of sugar composition on the performance of Lactobacillus mucilaginosus strains in modifying the profile of volatile compounds present in a sample.

[0277] [ Figure 4 ] shows that in Example 1, Proportional distribution of different volatile compounds in raw pea isolates grouped by sensory characteristics and fermented by different strains.

[0278] Example

[0279] Example 1: Process according to the invention starting from pea isolate

[0280] Strains and culture conditions :

[0281] - Inoculate a 10 mL pre-culture tube of the appropriate medium for the strain from the cryovial (frozen strain) with sugar.

[0282] - Incubate the tube in an oven without stirring at the optimal temperature for each strain,

[0283] Up to 15 hours to 24 hours.

[0284] - The pre-culture tube is then used to inoculate a 1 L flask or Erlenmeyer flask with 1% of the culture medium.

[0285] - Propagation is carried out at the optimal temperature for each strain for its optimal time.

[0286] - The growth parameters of the strains are summarized in Table 2 below:

[0287] Table 2

[0288]

[0289] (MRS = De Man, Rogosa and Sharpe agar; MW = Malt Wickerham agar)

[0290] Preparation of inoculum :

[0291] - At the end of the growth period, washes were applied to the different cultures in order to remove the culture medium.

[0292] - Centrifuge the culture at 4000G for 10 minutes at 4°C. Remove the supernatant and resuspend the pellet in physiological water.

[0293] -Then perform a new centrifugation and remove the supernatant.

[0294] - The strain was then resuspended in a volume of physiological water based on the calculated biomass so as to 8 CFU / mL inoculated test medium (thus in order to have 10 9 CFU / mL of inoculum).

[0295] Preparation of matrix (test medium) :

[0296] - The test medium or substrate consists of 10% pea isolate ( S85F) solution composition. Under stirring, 100g of pea isolate was added to sterile distilled water in a thin stream,

[0297] In order to obtain 900 mL of suspension.

[0298] - Add 100 mL of inoculum to this volume in order to obtain 1 L of suspension.

[0299] - The suspension was stirred for 30 min in order to ensure proper hydration and solubilization of the protein isolate.

[0300] Whole-cell biomass (abbreviated as WCB. Also called "whole-cell biocatalysis") treatment: The effect of the strain on the test medium with a short contact time (the principle is to use cells as a metabolic catalytic system with little or no cell growth):

[0301] - In preparation 10 9 CFU / mL inoculum, the test medium was inoculated with 100 mL of the inoculum in 900 mL of the test medium.

[0302] - Once the culture medium is inoculated, place the stoppered Pyrex flask in an oven at 30°C with stirring at 130 rpm for one hour.

[0303] - After treatment, the samples were spray dried to stop the action of the strain. Nebulization was performed at 160°C with 10% product feed and 100% suction.

[0304] - Samples collected in powder form were stored in sterile glass vials until the measurement of volatile compounds by SPME-GC-MS.

[0305] WCB Results - Interpretation

[0306] The results are shown in Figure 1 middle.

[0307] The effects on volatile compounds are presented as area per gram of sample (ie pea isolate) and averaged over three samples. Figure 1 The results depicted in are grouped by chemical family.

[0308] The results showed that the method of the present invention can change the processed pea isolate ( The volatile compound profile of S85F (S85F) was analyzed. In the obligate heterofermenters (Lactobacillus mucilaginosus fermentans and Lactobacillus mucilaginosus reuteri), the conversion of aldehydes to alcohols was very high. Lactobacillus mucilaginosus reuteri was also effective with ketones. This effect was also observed in Saccharomyces cerevisiae. Lactobacillus rhamnosus also converted aldehydes to carboxylic acids (not detected).

[0309] The conversion of aldehydes and ketones into other compounds (alcohols and / or carboxylic acids) reduces the undesirable "beany" or "vegetal" flavor. Indeed, aldehydes and ketones are the primary contributors, and their biotransformation products have less distinctive odors and higher perception thresholds. Consequently, these undesirable flavors are less or no longer perceived in the product. Reducing the amount of green-note aldehydes leads to improved sensory properties in the product by reducing the "vegetal" sensory attribute.

[0310] Chemical analyses were performed and are presented in Table 8 below:

[0311]

[0312]

[0313] It is thus observed that, using the method according to the invention:

[0314] -The production of lactic acid and ethanol is very low.

[0315] - pH and viability remained unchanged, indicating the absence of growth.

[0316] -DH remained unchanged, indicating that there was no hydrolysis.

[0317] A panel of five people tasted different samples:

[0318] - S85F pea isolate is described as bitter and vegetal

[0319] - Treatment with Lactobacillus rhamnosus LGG reduces the vegetative notes in favor of milky notes. This record indicates an interest in milky drinks.

[0320] - Treated with fermented Lactobacillus mucilaginosus, the vegetal notes disappear in favor of the umami notes.

[0321] - Treated with brewer's yeast, the vegetal notes disappear in favor of the cereal notes.

[0322] -Treatment with Gluconobacter oxydans removes the vegetal notes but leaves no room for any other flavors of interest.

[0323] Figure 4 Shown is the distribution of the proportions of various volatile compounds in the different samples produced by Example 1, grouped according to sensory characteristics.

[0324] The grouping by sensory characteristics was based on the literature, in particular the website www.thegoodscentscompany.com.

[0325] Therefore, the volatile compounds are grouped as follows:

[0326] - The "green" group will consist of: hexanal, heptanal, 2-hexenal, 2-heptenal,

[0327] (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)-.

[0328] - The "greasy" 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)-.

[0329] - The "herbal" group will consist of: 2-dodecenal, 1-hexanol, 3-octanone, and eucalyptol

[0330] - The "Other" group will consist of: 2,3-octanedione, octanoic acid and methyl ester

[0331] - The "earthy" group would consist of: 1-octen-3-ol, 2-octanone, 1-octen-3-one and 3-octen-2-one, (E)-

[0332] - The "bitter almond" group will be composed of: benzaldehyde.

[0333] - The "aldehyde" group will consist of: octanal, nonanal and decanal

[0334] - The "baking chocolate" group would consist of: butyraldehyde and 3-methyl-furan, 2-(1-pentenyl)-, (E)-

[0335] - The "Fermentation" group will consist of: Valeraldehyde, 1-pentanol, Decanoic acid, Methyl ester

[0336] - The "sulfur-containing" group would consist of: disulfide and dimethyl trisulfide

[0337] - The "fruity / floral" group would consist of: 2-pentanone, 1-nonanol, 1-hexanol, 2-ethyl

[0338] -, 2-nonanone, 2-decanone, 5-hepten-2-one, 6-methyl-, 2-n-butylfuran, 2(3H)-furanone, 5-butyldihydro, 2(3H)-furanone, 5-hexyldihydro-, phenylethanol, hexanoic acid, methyl ester, 3-nonen-2-one, 2-undecanone

[0339] - The "cheese" group will be composed of: 2-heptanone

[0340] - The "Solvents / Alcohols" group will consist of: 2-propanone, acetaldehyde, ethanol and furan, 2-ethyl-

[0341] therefore, Figure 4 can be interpreted as:

[0342] - In the "green" group, the profiles of Nutralys (untreated pea isolate) and Nutralys treated with Gluconobacter suboxydans were very similar and shared approximately 3 / 4 of the compounds.

[0343] Although the levels of these compounds were reduced, the overall sensory profile remained unchanged.

[0344] - In the "green" group, the profiles of Nutralys treated with S. cerevisiae and L. mucilaginosus were very similar and only shared about 50% of the compounds. The remainder, therefore, consisted of volatile compounds from other groups, providing a more complex profile.

[0345] - In the "green" group, the profiles of Nutralys treated with Lactobacillus reuteri and Lactobacillus rhamnosus were similar, with a complete disappearance of volatile compounds. Their sensory profiles were fundamentally different.

[0346] In conclusion and in contrast to patent EP 0 255 588 which teaches the use of Gluconobacter suboxydans, our method does more than just reduce the vegetal notes. The method according to the invention makes it possible to do this while producing a complex and unique organoleptic profile.

[0347] Due to the reduced contact time, no lactic acid is produced and the protein content remains unchanged, especially the degree of hydrolysis.

[0348] Example 2: Methods using strains of interest but using them in fermentation outside the scope of the present invention Law :

[0349] The purpose of this example is to demonstrate that long contact times between inoculum and isolate, typically 8 hours or 24 hours according to the disclosures of the prior art, as mainly described in the prior art for these strains, do not allow obtaining the same composition as according to the invention.

[0350] Obtaining inoculum :

[0351] culture medium :

[0352] - MRS (ready-to-use, Merck) for fermentation of Lactobacillus mucilaginosus, Lactobacillus mucilaginosus and Lactobacillus rhamnosus strains.

[0353] - YE glc (bacterial yeast extract, Difco 10 g / L + glucose 40 g / L) for Saccharomyces cerevisiae and Gluconobacter suboxydans strains.

[0354] nourish :

[0355] o In a 500 mL unbaffled Erlenmeyer flask containing 100 mL of culture medium for fermentation of Lactobacillus mucilaginosus, Lactobacillus mucilaginosus and Lactobacillus rhamnosus strains, and in an Erlenmeyer flask with 4 bottom baffles + 1 drop of defoamer containing 100 mL of culture medium for Saccharomyces cerevisiae and Gluconobacter suboxydans strains.

[0356] ○ Inoculation: 1%, use one tube of frozen stock solution (1 mL)

[0357] o Incubation: Anaerobic (bell jar) at 80 rpm at 37°C for 16 hours for Lactobacillus mucilaginosus, Lactobacillus mucilaginosus and Lactobacillus rhamnosus. Aerobic at 30°C for Gluconobacter suboxydans and at 150 rpm at 37°C for Saccharomyces cerevisiae for 16 hours

[0358] Biomass preparation: For all strains except Gluconobacter suboxydans, centrifuge the biomass from the Erlenmeyer flask at 3000 g for 10 minutes, which will require centrifugation at 10,000 g. Remove the supernatant and add the biomass to 10 mL of sterile physiological water at 4°C. (Due to the required inoculum, biomass washing is not necessary). Measure the OD at 600 nm and count the cell suspension.

[0359] At natural pH S85F 10% fermentation :

[0360] - equipment : 2L unbaffled conical flask × 11 sterile demineralized water flasks; about 6L

[0361] -program :

[0362] o Pre-incubate water flasks at 30°C and 37°C at 110 rpm.

[0363] ○50g S85F is placed in each conical flask

[0364] ○Add sterile water qsp 500g

[0365] ○ Allowed to rehydrate at various incubation temperatures tested 30 minutes.

[0366] - Erlenmeyer incubation parameters

[0367] ○Nutralys: Nutralys + Gluconobacter oxydans: 30°C, 110 rpm

[0368] Nutralys+ Saccharomyces cerevisiae: 37°C, 110 rpm

[0369] Nutralys+ Lactobacillus mucilaginosus, Lactobacillus mucilaginosus, and Lactobacillus rhamnosus: 37°C, 60 rpm + anaerobic bell jar

[0370] -Calculations used to implement 1.10 7 Inoculum volume of initial activity in CFU / mL

[0371] Correlation between OD and vitality (using Symbols represent):

[0372] ■Saccharomyces cerevisiae CBS 8066:

[0373] ■Gluconobacter oxydans ATCC 19357 (or ATCC 23773): OD

[0374] Lactobacillus rhamnosus GG LMG18243 (LGG): OD

[0375] ■ Fermented Lactobacillus mucilaginosus CNCM I-5802:

[0376] Lactobacillus reuteri DSM 17509:

[0377] ○For each strain, Erlenmeyer doubling (2 × 5 strains + 1 control)

[0378] ○ All test materials should be inoculated as early as in the morning

[0379] ○ At t0, take out about 5 mL of sample from one of the two conical flasks

[0380] o The following analyses were performed: pH, ethanol (Cedex Bio), lactate (Cedex Bio), viability (inoculum + t0), acetate, DH, protein content (N6.25), dry matter.

[0381] o Stop the Erlenmeyer control and transfer it to a flask for freezing.

[0382] o After 8 hours of fermentation, for each test strain, one of the two Erlenmeyer flasks was stopped.

[0383] o The following analyses were performed: pH, ethanol (Cedex Bio), lactate (Cedex Bio), viability (inoculum + t0), acetate, DH, protein content (N6.25), dry matter.

[0384] o After 24 hours of fermentation, for each test strain, the last Erlenmeyer flask was stopped.

[0385] The following analyses were performed: pH, ethanol (Cedex Bio), lactate (Cedex Bio), viability (inoculum+t0), acetate, DH, protein content (N6.25), dry matter.

[0386] The results are presented in Tables 3 and 4:

[0387] Table 3

[0388] Table 3: Ethanol / Lactate / pH / Activity Measurements

[0389] Table 4

[0390] Table 4: Dry matter measurement / DH / N x 6.25

[0391] In summary, the use of the strains of interest in methods outside the scope of the present invention (long contact time) produced isolates containing high levels of lactic acid. DH increased, particularly for L. reuteri, S. cerevisiae and Gluconobacter suboxydans, indicating protein hydrolysis.

[0392] Example 3: Demonstration of the importance of pre-WCB culture time :

[0393] The same protocol as in Example 1 was applied. However, with a volume of 250 mL and 25 g Conduct a test.

[0394] In this example the effect of the propagation time of a strain on its efficacy as a biocatalyst was investigated. Three propagation times for inoculum preparation were tested: cells were harvested after 5 hours, 10 hours or after 15 hours.

[0395] Two strains were tested: Lactobacillus fermentum (Lf) and Lactobacillus rhamnosus (Lr).

[0396] Results - Interpretation

[0397] The results are shown in Figure 2 middle.

[0398] Figure 2 Shown in Effect of cultivation time of Lactobacillus fermentans (Lf) and Lactobacillus rhamnosus (Lr) strains before F85F exposure on their efficacy in improving sensory quality.

[0399] Two situations were observed:

[0400] - For Lactobacillus mucilaginosus (obligate heterofermentative), the minimum incubation time to obtain optimal biocatalytic activity was 10 hours.

[0401] - For Lactobacillus rhamnosus (facultative heterofermentative), the minimum incubation time is 15 hours.

[0402] Therefore, different strains require different culture times to achieve maximum effect. A minimum propagation time of 10 hours is preferred to obtain the maximum technical effect of the present invention.

[0403] Example 4: Demonstration of the importance of sugar concentration in the WCB step :

[0404] This embodiment uses the same scheme as embodiment 1.

[0405] The focus here is on the comparison of research impact:

[0406] - selected strains (Lactobacillus muciferum),

[0407] - Replacement of pea protein isolate on a model matrix (volatile compounds causing undesirable flavor, plus buffer) S85F)

[0408] - With sugar and without sugar.

[0409] Comparison of three sugar contents :

[0410] -0g / L,

[0411] -0.5g / L (10% solution Sugar content, ≈ during the process)

[0412] -5g / L( sugar content).

[0413] Make a 1.6 ppm volatile compound solution using:

[0414] -hexanal,

[0415] -nonanal,

[0416] -2-nonenal,

[0417] -benzaldehyde,

[0418] -3-methylbutanal,

[0419] -1-octen-3-ol,

[0420] -3-octen-2-one, and

[0421] -2,5-dimethylpyrazine.

[0422] From this solution, three solutions with three sugar contents were prepared, as shown below.

[0423] - 1.6ppm sugar-free tracer

[0424] -Add phosphate buffer (pH 6.5 0.1M), 4.9g / L K2HPO4 and 9.9g / L KH2PO4

[0425] -6 x 9mL tubes

[0426] -Sterile filtration (0.22μm)

[0427] - 1.6ppm tracer with 0.5g sugar

[0428] -Add phosphate buffer (pH 6.5 0.1M), 4.9g / L K2HPO4 and 9.9g / L KH2PO4

[0429] -Added sugars:

[0430] Table 5

[0431] glucose fructose Mannose Galactose Xylose Arabinose poisoning Raffinose 0.15g / L 0.058g / L 0.030g / L 0.12g / L 0.030g / L 0.19g / L 0.003g / L

[0432] □6×9mL tubes

[0433] □Sterile filtration (0.22μm)

[0434] 1.6ppm tracer with 5g sugar

[0435] -Add phosphate buffer (pH 6.5 0.1M), 4.9g / L K2HPO4 and 9.9g / L KH2PO4

[0436] -5g added sugar:

[0437] Table 6

[0438] glucose fructose Mannose Galactose Xylose Arabinose poisoning Raffinose 1.5g / L 0.58g / L 0.30g / L 1.20g / L 0.30g / L 1.90g / L 0.03g / L

[0439] -6 x 9mL tubes

[0440] -Sterile filtration (0.22μm)

[0441] touch

[0442] - The same protocol as the WCB procedure applies.

[0443] Results and Interpretation

[0444] The test was performed in triplicate using three different pre-cultures. The effect of Lactobacillus mucilaginosus fermenting on the tracer based on sugar content is shown in the figure below.

[0445] The results are shown in Figure 3 and Table 7.

[0446] Table 7

[0447]

[0448] Figure 3 and Table 7 show the effect of sugar composition on the performance of Lactobacillus mucilaginosus strains in modifying the profile of volatile compounds present in the samples.

[0449] Example 5: Demonstration of the importance of cell concentration before treatment and the possibility of using lyophilized inoculum :

[0450] This example uses the same solution as Example 1, except for three points:

[0451] - Two strains were studied: Lactobacillus fermentum and Lactobacillus rhamnosus.

[0452] - Alternatively, the frozen strain was introduced into the lyophilized strain. Before use, the strain was dispersed in a solution containing 1 g / L glucose and 10 g / L pea isolate (such as

[0453] The inocula were then used as in Example 1, except for their concentrations (see below).

[0454] - After resuspending the strain in a certain volume of water, the 10 8 CFU / mL, 10 7 CFU / mL or 10 4 CFU / mL of inoculum required for the amount of water.

[0455] The following facts can be observed:

[0456] -Use 10 4 The results obtained for CFU / mL were very poor: aldehydes, especially hexanal, were not consumed.

[0457] So from a flavor perspective, the protein composition remains unchanged.

[0458] From this perspective, whether fresh, frozen or freeze-dried strains are used, 8 CFU / mL and 10 7 The results obtained for CFU / mL were also good. Therefore, lyophilization is an excellent means of preserving strains before processing.

Claims

1. A method for improving the sensory properties of a material rich in vegetable protein, the method comprising the following steps:

1. Suspending a material rich in vegetable protein in a preferably aqueous solvent in order to obtain a material having a content of between 5% and 20%, preferably between 7.5% and 15%, even more preferably between 8%. Suspensions with a dry matter content between 12%; 2. Add one or more microbial strains to the suspension of step 1 to obtain With 1.10 5 cfu / mL and 1.10 10 cfu / mL, preferably 1.10 7 cfu / mL With 1.10 9 cfu / mL, preferably between 5.10 7 cfu / mL and 5.10 8 cfu / mL, wherein the microbial strain is selected from a lactic acid bacteria strain, a yeast strain or a mixture thereof, and the strain is preferably one or more lactic acid bacteria. strain; 3. Incubate the suspension from step 2 at a temperature between 20°C and 40°C for 30 minutes. between 1 hour and 120 minutes, preferably between 45 minutes and 75 minutes, or even better The duration is preferably between 50 and 70 minutes.

2. The method according to claim 1, characterized in that The vegetable protein-rich material of step 1 is in the form of a powder containing vegetable protein in an amount greater than 1 wt%, preferably between 70 wt% and 90 wt%, relative to the weight of the powder.

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

4. The method according to any one of claims 1 to 3, characterized in that The plant protein-rich material of step 1 is derived from legumes, preferably from legumes selected from the group consisting of peas and broad beans, more preferably from peas.

5. The method according to any one of claims 1 to 4, characterized in that Before carrying out step 2, the pH of said suspension of vegetable protein-rich material of step 1 is modified so as to be between 5.0 and 7.5, preferentially between 6.5 and 7.5; preferentially so as to be equal to 7.

0.

6. The method according to claims 1 to 5, characterized in that The sugar content of said suspension of vegetable protein-rich material of step 1 is modified so as to contain between 1% and 10% sugar, preferentially between 2% and 8% sugar, even more preferentially between 3% and 6% sugar.

7. The method according to claims 1 to 6, characterized in that The one or more strains of step 2 are selected from Limosilactobacillus fermentum, Limosilactobacillus reuteri, Lactobacillus rhamnosus or Saccharomyces cerevisiae, or strains that preferentially ferment Limosilactobacillus fermentum, Limosilactobacillus reuteri or Lactobacillus rhamnosus.

8. The method according to claims 1 to 7, characterized in that The one or more strains used in step 2 have been cultured according to a method comprising: i. inoculating a solid medium, such as agar, with a sample of the strain; ii. pre-culturing the strain by incubating the inoculated solid medium of step 1 in an oven without stirring, preferably for between 10 hours and 24 hours; iii. multiplying the strain by inoculating a liquid culture medium with a sample of the strain from the pre-culture of step ii and culturing the strain in the liquid culture medium.

9. The method according to claim 8, characterized in that The incubation time of step iii is between 10 hours and 20 hours.

10. The method according to claims 1 to 9, characterized in that Step 3 is followed by step 4, which includes the following sub-steps: 4a. Optionally, modifying the pH of the suspension obtained at the end of step 3 to a pH between 5 and 9, preferentially to a pH between 6 and 9, preferentially to a pH of 7, preferentially by adding soda ash or lime to said suspension; 4b. heat treating the suspension of step 3 or step 4a at between 100°C and 160°C for 0.1 to 1 second, 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 the isolate.

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

12. The plant protein isolate according to claim 11, characterized in that The degree of hydrolysis (or DH) of the vegetable protein isolate is less than 10%, preferably less than 5%.

13. The plant protein isolate according to any one of claims 11 to 12, characterized in that The protein content of the vegetable protein isolate 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 weight of the dry matter of the isolate.

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

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