Plant protein isolates extracted and enhanced via microbial strains

Abstract

The invention relates to a method for extracting a plant protein, comprising the following steps: 1. Suspending a plant powder in a preferential aqueous solvent in order to obtain a plant powder suspension; 2a, adding a strain into the plant powder suspension in the step 1; 2b. Incubating the inoculated suspension of step 2a such that the strain acidifies the inoculated suspension until a protein precipitate is formed; 3. Separating the protein precipitate obtained at the end of step 2b; and the invention relates to a plant protein isolate obtainable by the method of the invention.

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 proteins 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 varieties. These seeds are non-GMO and do not require a de-oiling step using solvents.

[0008] Pea protein (primarily vicilin) has 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 directed to 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 flour. This flour 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 several disadvantages. The first is the use of acid-base reagents, which are expensive and require investment for storage and processing. They are also harmful to humans, as contact with them without adequate protection can cause serious damage. A second disadvantage is that the molecular integrity of the isolate obtained is affected, and its organoleptic properties are also affected. It is well known that heating under acidic conditions can significantly modify protein molecules and lead to the appearance of organic volatile compounds produced by lipid degradation. Examples of such compounds are hexanal or methyl mercaptan.

[0011] In the present invention, the present invention relates to the method for the extraction of the raw material of the present invention.Therefore, this is common practice, and in the field of method improvement, many works are being carried out at present, to replace this isoelectric precipitation step under the situation of heating or not heating, or even to increase extra method step to overcome these undesirable influences.Unfortunately, the first solution needs to invest in membrane filtration technology, which is expensive and complicated to implement due to the significant blocking of filter medium.The second solution also has many restrictions, not only because in already complicated method, new steps are implemented.Before the extraction method that constitutes the basis of patent EP 3 071 046, the fermentation of leguminous plant seeds can be mentioned in particular.Though the final result is useful, this method relates to the large amount of fermentation that continues to exceed 24 hours, and produces the excessive residual water that needs to be handled.

[0012] A recent alternative approach is to use bacterial strains to replace the acid-base reagents in the precipitation step, as described in the article by Emkani et al. 2021 (Emkani et al., Foods, 10, 549, 2021: “Pea Protein Extraction Assisted by Lactic Fermentation: Impact on Protein Profile and Thermal Properties”). Although this method seems to be beneficial, it also has two disadvantages: hydrolysis or even consumption of protein by the strain, and a fermentation time of about 5 to 6 hours that is not easily compatible with industrial methods. The article “Protein composition and nutritional aspects of pea protein fractions obtained by amodified isoelectric precipitation method using fermentation” (Emkani et al., Frontiers in Nutrition, Vol. 10, 2023) therefore proves that “protein is degraded into small peptides and amino acids by LAB, which are dissolved in the soluble fraction (albumin) as confirmed by size exclusion chromatography (SEC-HPLC)”.

[0013] The applicant deserves credit for having worked in this field and discovered a group of strains that achieve rapid acidification without hydrolysis (i.e. without consuming protein), and has also developed a method for using this group of strains, with the aim of obtaining protein isolates whose functional and organoleptic characteristics 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.

[0014] The present invention will be better understood in the following description portion of the present application.

[0015] General Description

[0016] The present invention relates to a method for extracting plant protein, which comprises the following steps:

[0017] 1. suspending the plant powder in a preferred aqueous solvent to obtain a plant powder suspension;

[0018] 2a. Adding a strain to the plant powder suspension of step 1 to form an inoculum suspension, the strain being selected from the group consisting of: Lactobacillus fermentum, Lactobacillus salivarius, Lactobacillus delbrueckii subsp Jakobsenii, Lactobacillus mucosae, Streptococcus salivarius and Enterococcus lactis; the strain is preferably a Lactobacillus fermentum strain; even more preferably a strain deposited at the CNCM under the number CNCM I-5802;

[0019] 2b. incubating the inoculated suspension of step 2a so that the strain acidifies the inoculated suspension until a protein precipitate is formed;

[0020] 3. Isolate the protein precipitate obtained at the end of step 2b.

[0021] In one embodiment, the process according to the invention is characterized in that the vegetable meal of step 1 is in the form of a vegetable powder containing an amount of protein greater than 1% by weight, preferentially between 15% and 30% by weight relative to the weight of the powder.

[0022] In one embodiment, the process according to the invention is characterized in that the vegetable meal of step 1 is an isolate or a concentrate, preferentially an isolate having a protein content greater than 80% by dry weight relative to the dry matter of the isolate.

[0023] In one embodiment, the process according to the invention is characterized in that the plant meal of step 1 is a leguminous plant meal, preferentially characterized in that the plant meal is a leguminous plant meal chosen from the group consisting of peas and beans, more preferentially characterized in that the plant meal is pea meal.

[0024] In one embodiment, the method according to the invention is characterized in that the incubation temperature of the suspension of step 2b is between 30°C and 50°C, preferentially between 35°C and 45°C.

[0025] In one embodiment, the process according to the invention is characterized in that, before carrying out step 2a, the pH of the plant powder suspension of step 1 is adjusted to a pH between 6.5 and 7.5, preferably equal to 7.0.

[0026] In one embodiment, the method according to the invention is characterized in that the addition of the strain in step 2a is carried out so as to obtain a strain concentration of 0.1×10 9 to 1×109 The suspension was inoculated with a colony forming unit (CFU).

[0027] In one embodiment, the method according to the invention is characterized in that the incubation of step 2b is carried out until the inoculated suspension has a pH between 4 and 5, preferentially a pH of 5.0.

[0028] In one embodiment, the process according to the invention is characterized in that if, at the end of step 2b, the pH of the inoculated suspension is not between 4 and 5, step 2b comprises, at the end of step 2b, a sub-step 2b' of acidifying the inoculated suspension by adding an acid, such that the inoculated suspension obtained at the end of step 2b has a pH between 4 and 5, preferably between 4.5 and 5.

[0029] In one embodiment, the process according to the invention is characterized in that step 2b comprises, at the end of step 2b, a sub-step 2b" of additional heating in order to increase the flocculation yield, this sub-step 2b" consisting in bringing the plant powder suspension of step 2b to a temperature comprised between 45°C and 85°C.

[0030] In one embodiment, the method according to the invention is characterized in that step 3 comprises the following steps:

[0031] 3a. The pH of the inoculated suspension obtained at the end of step 2b is adjusted to a pH of 6 to 9, preferably a pH of 7, preferably by adding soda or lime to the suspension;

[0032] 3b. heat treating the suspension of step 3a at 100°C to 160°C for 0.1 to 1 second, and

[0033] 3c. Drying the plant powder suspension of step 3b by means of a sprayer until a plant powder isolate having a dry matter content greater than 95% is obtained.

[0034] The present invention also relates to a vegetable protein isolate obtainable by the process of the invention.

[0035] In one embodiment, the vegetable protein isolate according to the invention is characterized in that its degree of hydrolysis (or DH) is less than 10%, preferably less than 5.

[0036] In one embodiment, the vegetable protein isolate according to the invention is characterized in that the methyl mercaptan content is reduced by more than 25%, preferentially by more than 50%, relative to an isolate obtained by acidifying the plant powder suspension by adding hydrochloric acid alone.

[0037] In one embodiment, the vegetable protein isolate according to the invention is characterized in that its protein content 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.

[0038] The present invention also relates to the use of an isolate according to the invention or an isolate obtained by a process according to the invention for industrial applications including food, nutraceutical and pharmaceutical applications.

[0039] The invention also relates to the microbial strain deposited at the CNCM under the number CNCM 1-5802.

[0040] The invention also relates to a vegetable protein isolate, characterized in that its hexanal content is less than 6000 μg / kg and its methyl mercaptan content is less than 100 μg / kg.

[0041] The present invention will be better understood by reading the following detailed description. DETAILED DESCRIPTION

[0042] The present invention relates to the microbial strain deposited at the CNCM under the number CNCM 1-5802.

[0043] This particular strain has been identified as belonging to the genus Lactobacillus fermentum. As will be demonstrated in the Examples section, this strain stands out in a particular way by making it possible to quickly and effectively acidify culture media containing vegetable proteins, preferentially pea proteins.

[0044] Strain CNCM 1-5802 will be used primarily in plant protein extraction methods, but also in any other activity requiring rapid acidification with little protein hydrolysis. These applications will be reviewed in further detail and non-exhaustively in this specification.

[0045] The present invention therefore relates to a method for extracting plant protein, comprising the following steps:

[0046] 1. suspending the plant powder in a preferred aqueous solvent to obtain a plant powder suspension;

[0047] 2a. Adding a strain to the plant powder suspension of step 1 to form an inoculated suspension, the strain being selected from the group consisting of: Lactobacillus fermentum, Lactobacillus salivarius, Lactobacillus delbrueckii subsp. Jacobsen, Lactobacillus mucosae, Streptococcus salivarius and Enterococcus lactis; the strain is preferably a Lactobacillus fermentum strain; even more preferably a strain deposited in CNCM under the number CNCM I-5802;

[0048] 2b. incubating the inoculated suspension of step 2a so that the strain acidifies the inoculated suspension until a protein precipitate is formed;

[0049] 3. Isolate the protein precipitate obtained at the end of step 2b.

[0050] The first step of the method therefore consists in using plant powder.

[0051] For the purposes of the present invention, “vegetable flour” means any flour of vegetable origin containing protein in an amount greater than 1% by weight, preferentially between 15% and 30% by weight relative to the weight of the flour.

[0052] Specifically, for the purposes of the present invention, this will mean flour obtained by grinding plant seeds. These seeds may have undergone pretreatments such as cleaning, sieving, thermal heating, soaking or bleaching steps, or sieving (e.g., to separate the seeds from stones). Preferably, if bleaching is performed, the heat treatment regime will be at 80° C. for 3 minutes.

[0053] Plant seeds consist of a structure that contains and protects the plant embryo. It is usually contained within a fruit, allowing it to spread, or protected by a shell, often called a husk. These plants are particularly selected from legumes and cereals.

[0054] For the purposes of this invention, "legumes" refers to the dicotyledonous plant family of the order Fabales. 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, comprising over 19,500 species. Several legumes are important crop plants, including beans, peas, faba beans, lupines, common beans, chickpeas, peanuts, cultivated lentils, cultivated alfalfa, various clover species, broad beans, carob, and liquorice.

[0055] Preferably, the leguminous plant is selected from peas and beans, even more preferably peas.

[0056] The term "pea" is considered herein to be used in its most widely accepted sense and specifically includes all varieties of "smooth pea" and "wrinkled pea" and all mutants of "smooth pea" and "wrinkled pea", regardless of the use (human food, animal feed and / or other uses) for which the variety is typically used. The term "pea" in this application includes pea varieties belonging to the genus Pisum, more specifically Pisum sativum and Pisum aestivum. The mutants are particularly those referred to as "mutant r", "mutant rb", "mutant rug 3", "mutant rug 4", "mutant rug 5" and "mutant lam", as described in C-LHEYDLEY et al., "Developing novel peastarches", Proceedings of the Symposium of the Industrial Biochemistry and Biotechnology Group of the Biochemical Society, 1996, pp. 77-87.

[0057] The term "faba bean" is intended to refer to an annual plant of the species Vicia faba, belonging to the legume family of the Fabaceae family, Faboideae subfamily, and Fabeae tribe. A distinction is made between minor and major varieties. For the purposes of the present invention, both wild-type varieties and those obtained through genetic engineering or cultivar selection are excellent sources.

[0058] Therefore, the vegetable powder will be preferentially reduced to a powder with a finer particle size. All technologies known from the prior art are compatible with the method according to the invention. For example, a chopper may be mentioned. Specific, particularly suitable examples of such a chopper are, for example, SM300 sold, or for example by Stone mill for sale.

[0059] The powder obtained in this way is suspended in a solvent. This solvent is not intended to completely dissolve all the same dry matter, but this is entirely conceivable. In a preferred embodiment, the conditions under which the powder is suspended are such that non-protein compounds are insoluble, in order to remove them and thus pre-enrich the solution in proteins.

[0060] Preferably, the solvent is water. The latter may, however, contain additives, for example compounds which facilitate solubility.

[0061] Preferably, the pH of the aqueous solvent is adjusted to 8 to 10, preferably to 9. Any alkaline agent such as soda, lime is possible, but potash is preferred. The temperature is preferably adjusted to 2° C. to 30° C., preferably 10° C. to 30° C., preferably 15° C. to 25° C., and even more preferably to 20° C. This temperature is regulated throughout the extraction reaction.

[0062] The powder is diluted in a preferably aqueous solvent to obtain a suspension of 5% to 25%, preferably 5% to 15%, preferably 7% to 13%, even more preferably 9% to 11%, and most preferably 10%, the percentages being expressed as the weight of the powder relative to the total weight of the water / powder suspension. The suspension is stirred using any means known to those skilled in the art, such as a drum equipped with a stirrer, a paddle, a marine propeller, or any other device that allows for effective stirring. The extraction time, preferably while stirring, is 5 to 25 minutes, preferably 10 to 20 minutes, even more preferably 15 minutes.

[0063] Preferably, the suspension is then centrifuged to remove insoluble non-protein compounds, such as starch and / or internal fibers, also known as pulp in the case of legumes.

[0064] In an alternative embodiment, the plant seed meal is a protein-rich material. "Protein-rich material" means any material in powder or flocculent form that contains at least 25% protein by dry weight relative to dry matter. Examples include concentrates, isolates, and seeds, in a non-limiting manner. Preferably, the protein-rich material is in powder form.

[0065] Preferably, the pea protein-rich material used in step 1 is an isolate, i.e., its protein content is greater than 80% by dry weight relative to the dry matter. The use of a concentrate (protein content of 50% to 80% by dry weight relative to the dry matter) or even a flour (protein content of less than 50% by dry weight relative to the dry matter) is also conceivable, but an isolate is preferred.

[0066] Pea protein-rich materials are readily obtained using conventional methods well known to those skilled in the art. For example, the methods described in the applicant's patent applications EP 1 909 593 or FR 2018052261 are mentioned. The basic principles of these methods (suspension of soy flour in water by wet or dry milling, removal of insoluble fractions such as starch and internal fibers by centrifugation, isoelectric precipitation of the protein of interest) are now commonplace and readily yield suitable proteins.

[0067] Regardless of the method used to obtain the vegetable protein suspension (e.g. simply by adding water to pea protein isolate, or starting with pea flour and removing the internal fiber and starch), the final dry matter of the suspension (also called "crude extract") will preferentially be between 3% and 9%, thus including the values 3%, 4%, 5%, 6%, 7%, 8% and 9%. The percentages are expressed in grams of dry matter per 100 grams of total weight of the protein suspension.

[0068] The second step of the method according to the present invention comprises at least:

[0069] 2a. Adding a strain to the plant powder suspension of step 1 to form an inoculated suspension, the strain being selected from the group consisting of: Lactobacillus fermentum, Lactobacillus salivarius, Lactobacillus delbrueckii subsp. Jacobsen, Lactobacillus mucosae, Streptococcus salivarius and Enterococcus lactis; the strain is preferably a Lactobacillus fermentum strain; even more preferably a strain deposited in CNCM under the number CNCM I-5802;

[0070] 2b. Incubate the inoculated suspension of step 2a, allowing the strain to acidify the inoculated suspension until a protein precipitate forms.

[0071] The second step therefore consists in acidifying the pH using a strain chosen from Lactobacillus fermentum, Lactobacillus salivarius, Lactobacillus delbrueckii subsp. jacobsenii, Lactobacillus mucosae, Streptococcus salivarius and Enterococcus lactis; preferentially a Lactobacillus fermentum strain; even more preferentially a strain CNCM 1-5802.

[0072] Preferably, the temperature of the protein suspension is regulated to 30°C to 50°C, preferably 35°C to 45°C, thus including the values of 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C and 45°C;

[0073] Preferably, the initial pH is adjusted to 6.5 to 7.5; preferably to 7.0; thus including values of 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4 and 7.5. To this end, those skilled in the art will use any well-known equipment and reagents.

[0074] A strain selected from the group consisting of Lactobacillus fermentum, Lactobacillus salivarius, Lactobacillus delbrueckii subsp. Jacobsenii, Lactobacillus mucosae, Streptococcus salivarius and Enterococcus lactis; preferably Lactobacillus fermentum; even more preferably a strain of strain CNCM I-5802; then preferably at 0.1×10 9 to 1×10 9 The concentration of CFU (colony forming units) was introduced.

[0075] Preferably, the strain inoculum is prepared as follows, hereinafter referred to as "the method for preparing inoculum A":

[0076] -Store the strain in frozen beads

[0077] - Inoculate a 2 L Erlenmeyer flask with 500 mL of MRS medium and 1 bead from the strain frozen beads.

[0078] - in an anaerobic incubator at a temperature of 37°C + / - 1°C and under stirring at 80 rpm,

[0079] Incubate for 18 to 24 hours

[0080] - Transfer the contents of the Erlenmeyer flask to a centrifuge container

[0081] - Centrifuge at 3000g for 10 minutes

[0082] - Remove the supernatant

[0083] - Resuspend the pellet with physiological water

[0084] - Repeat the centrifugation / washing step twice

[0085] - After the last centrifugation, the pellet was recovered in 10 mL of physiological water

[0086] - Store the resulting suspension on ice or at 4°C until use

[0087] Generate a correlation line between OD 600nm and CFU / ml. This is achieved by preparing several 10-fold dilutions of the bacterial suspension, then measuring the OD 600nm and counting the CFU / ml. Plot the curve CFU / ml = f(OD 600nm). Linear regression can thus be used to obtain a correlation coefficient between OD 600nm and the number of CFU / ml, allowing one to be calculated from the other.

[0088] - Measure the OD 600nm of the suspension stored at 4°C using a spectrophotometer (if dilution is required to obtain measurements, dilute the cell suspension in 0.3% Tween, as biomass tends to stick to the walls of the dilution tube)

[0089] - Determine the volume of inoculum to be introduced into a given volume of crude protein extract in order to achieve 0.1 × 10 9 to 1×10 9 The concentration of CFU (colony forming units).

[0090] Preferably, and to give an example, a suspension of Lactobacillus fermentum strain CNCM 1-5802 with an OD 600nm of 1 corresponds to 3.0×10 8 The concentration of CFU / mL.

[0091] After the bacterial inoculum is introduced into the crude protein extract, stirring is performed using any equipment well known to those skilled in the art, including an agitator shaft equipped with a marine propeller. The stirring speed is preferentially 200 rpm to 400 rpm, preferentially 250 rpm to 350 rpm.

[0092] The pH will quickly become more acidic. Use a suitable probe to monitor changes in pH. The maximum acidification rate (expressed as Vm) expressed in pH units / minute is typically -0.030 to -0.020 pH units / minute, preferably -0.027 to -0.022 pH units / minute. If the pH remains unchanged (no acidification), or if the acidification rate is less than -0.020 pH units / minute, then an additional amount of fermentate may be added to overcome the latter's poor acidification capacity.

[0093] The target pH is the so-called isoelectric pH of the protein to be flocculated. For example, for a vicilin fraction, the pH is 5. This can vary depending on the protein being targeted. Therefore, this natural acidification leads to protein flocculation and coagulation.

[0094] Preferentially, an additional amount of acid may be added at the end of step 2b (sub-step 2b') so that the acidification can be completed, for example from pH 5 to pH 4.5.

[0095] Preferably, additional heating may be performed, preferably at the end of step 2b, in order to increase the flocculation yield (sub-step 2b"). In the present invention, the term "flocculation" means that a portion of the proteins present in solution are insoluble, so that they can be extracted. "Flocculation" and "precipitation" are used interchangeably herein. The heating temperature is between 45°C and 85°C, thus including the values of 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, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C and 85°C.

[0096] When the protein flocs obtained are of the desired quality and amount, they are conventionally separated from the rest of the aqueous suspension by static or dynamic decanting (plate centrifuge, horizontal decanter, etc.).

[0097] The flocculent thus obtained can be used as such or can be subjected to one or more separate process steps, including separate steps of purification (e.g. by chromatography, ultrafiltration), concentration (e.g. evaporation, drying), addition of chemical reagents (e.g. to adjust the pH) / enzymatic reagents (e.g. proteases) and / or heat treatment (e.g. pasteurization, HTST, sterilization).

[0098] A preferred example of post-treatment consists of: 1) adjusting the pH to 6 to 9, preferably to 7, preferably using soda or lime; 2) heat treatment between 100°C and 160°C for 0.1 second to 1 second; and 3) drying using a sprayer to a dry matter content greater than 90%, preferably greater than 95%.

[0099] The present invention also relates to a plant protein isolate obtained or obtainable by isoelectric precipitation using a strain selected from the group consisting of Lactobacillus fermentum, Lactobacillus salivarius, Lactobacillus delbrueckii subsp. jacobsenii, Lactobacillus mucosae, Streptococcus salivarius and Enterococcus lactis; the strain is preferably a Lactobacillus fermentum strain; even more preferably the strain CNCM I-5802.

[0100] This isolate, obtained by isoelectric precipitation using bacterial acidification, is related to the prior art, including in particular the article by Emkani et al. (Emkani et al., Foods, 10, 549, 2021), which is characterized in that the degree of hydrolysis of the isolate remains unchanged. Without wishing to be bound by a particular theory, it is the choice of strain and its use that makes it possible to achieve this unique result, which is unparalleled to date to our knowledge. The absence of proteases, the presence of reduced activity and / or the metabolic acidification rate combine to achieve this result.

[0101] The vegetable protein isolate according to the invention is preferably characterized in that its degree of hydrolysis (or DH) is less than 10%, preferentially less than 5.

[0102] Isolates according to the present invention preferably have a degree of hydrolysis (or DH) of less than 10%, preferentially less than 5%, including DH of 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% and 1%.

[0103] 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.

[0104] Particular preference is given to the following test for measuring the degree of hydrolysis. Its principle consists firstly in determining the amino nitrogen content (free NH2 functional groups) of a protein sample (preferably using the MEGAZYME kit (reference K-PANOPA)), then in determining the protein nitrogen content (total nitrogen, therefore including free and bound NH2 functional groups) of the sample, and finally in calculating the degree of hydrolysis, which is obtained by the ratio of these two measured values.

[0105] Determination of amino nitrogen content:

[0106] 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.

[0107] 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.

[0108] 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.

[0109] Prepare blanks, standards, and samples directly in the cuvettes of the spectrophotometer under the following conditions:

[0110] -Blank: Add 3.00ml of solution 1 and 50μl of distilled water

[0111] - Standard: Add 3.00 ml of solution 1 and 50 μl of bottle 3 of the Megazyme kit

[0112] - Sample: Add 3.00 ml of Solution 1 and 50 μl of Sample Preparation Solution.

[0113] 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).

[0114] 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.

[0115] The contents of each cuvette were mixed and then placed in the dark for approximately 20 minutes.

[0116] Then obtain absorbance measurements A2 of the blank, standards, and samples from the spectrophotometer at 340 nm.

[0117] The free amino nitrogen content, expressed as a weight percentage relative to the weight of the product, is given by the formula:

[0118] [Formula 1]

[0119]

[0120] [Formula 2]

[0121]

[0122] in:

[0123] ΔAsam=Asam2–Asam1

[0124] ΔAblk=Ablk2–Ablk1

[0125] Asam2 = absorbance of the sample after adding solution 2

[0126] Asam1 = absorbance of the sample after adding solution 1

[0127] Ablk2 = absorbance of blank after adding solution 2

[0128] Ablk1 = absorbance of blank after adding solution 1

[0129] V = volume of the flask

[0130] m = weight of the sample (in g)

[0131] 6803 = Extinction coefficient of isoindole derivatives at 340 nm (in 1.mol -1 .cm -1 as units).

[0132] 14.01 = molar mass of nitrogen (in g.mol -1 (unit)

[0133] 3.15 = Final volume in the cuvette (in ml)

[0134] 0.05 = Sample in the cuvette (in ml)

[0135] Determination of protein nitrogen content:

[0136] 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.

[0137] Calculation of degree of hydrolysis:

[0138] The degree of hydrolysis (DH) was calculated using the following formula:

[0139] [Formula 3]

[0140]

[0141] The isolate obtained according to the method of the present invention is also characterized by a reduced methyl mercaptan content. As illustrated in this application, this compound is synthesized by degradation of sulfur-containing amino acids during conventional acidification (e.g., with hydrochloric acid). When the isolate is used to prepare ready-to-drink beverages or to prepare wet-extruded strips intended for the production of artificial meat, the compound is present at ppb levels sufficient to produce a flavor known as "rancid egg" or "hydrogen sulfide." Without wishing to be bound by a particular theory, these methods generate a large amount of heat, resulting in the appearance of large amounts of methyl mercaptan-derived degradation compounds. The process according to the invention preferentially makes it possible to obtain an isolate having a methyl mercaptan content reduced by more than 50%, preferentially by more than 25%, these reduction values being potentially 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%. The process according to the invention preferentially makes it possible to obtain an isolate having a methyl mercaptan content of less than 0.1 ppb, i.e. an isolate comprising less than 0.1 μg of methyl mercaptan / kg of isolate. Preferentially, the process according to the invention makes it possible to obtain an isolate having a methyl mercaptan content of less than 0.01 ppb, i.e. an isolate comprising less than 0.01 μg of methyl mercaptan / kg of isolate. More preferentially, the process according to the invention makes it possible to obtain an isolate having a methyl mercaptan content of less than 0.001 ppb, or in other words a methyl mercaptan content of less than 1 ppt (parts per trillion; 1 ppt=1 ng / kg), i.e. an isolate comprising less than 0.01 μg of methyl mercaptan / kg of isolate.

[0142] The vegetable protein isolate according to the invention is characterized in that its protein content (expressed relative to its total substance dry matter) is preferably 80% to 95%, preferentially 82% to 92%, preferentially 84% to 90%, preferentially 84% to 88%.

[0143] The invention also relates to a vegetable protein isolate, characterized in that its hexanal content is less than 6000 μg / kg and its methyl mercaptan content is less than 100 μg / kg.

[0144] According to the present invention, "hexanal" (or hexanaldehyde) means an organic compound from the aldehyde family with the empirical formula C6H12O, ie an isomer of hexanone. Its CAS number is 66-25-1.

[0145] According to the present invention, "methyl mercaptan" (or methyl mercaptan) refers to an organic sulfur compound having the chemical formula CH3SH. It is a colorless gas from the thiol family with an odor reminiscent of rancid cabbage. Its CAS number is 74-93-1.

[0146] The influence of volatile compounds, especially hexanal, on the aroma characteristics of pea protein has been known for decades. Although hexanal is important, it is also important to control other volatile compounds. Recently, methyl mercaptan has been highlighted as the main important compound producing sulfur aroma (see "Characterization of odor-active compounds of various pea preparations by GC-MS, GC-O, and their correlation with sensory attributes", Zhogoleva & al., Future Foods, Vol. 8, available online in June 2023).

[0147] These compounds are routinely determined using gas chromatography equipped with a mass spectrophotometer. Many protocols exist, and those skilled in the art will know how to find and adapt them to quantify these compounds. A particularly preferred protocol is described in paragraph 168 of this application.

[0148] The hexanal content is less than 6000 μg / kg, preferably less than 5500 μg / kg, preferably less than 5000 μg / kg, preferably less than 4500 μg / kg, preferably 4000 μg / kg, preferably 3500 μg / kg.

[0149] The methyl mercaptan content is less than 120 μg / kg, preferably less than 110 μg / kg, preferably less than 100 μg / kg, preferably less than 90 μg / kg, preferably 80 μg / kg, preferably 70 μg / kg, preferably 65 μg / kg.Therefore, the methyl mercaptan content can be 119μg / kg, 118μg / kg, 117μg / kg, 116μg / kg, 115μg / kg, 114μg / kg, 113μg / kg, 112μg / kg, 111μg / kg, 110μg / kg, 109μg / kg, 108μg / kg, 107μg / kg, 106μg / kg, 105μg / kg, 104μg / kg, 103μg / kg, 102μg / kg, 101μg / kg, 100μg / kg, 99μg / kg, 98μg / kg, 97μg / kg, 96μg / kg, 95μg / kg, 94μg / kg, 93μg / kg, 92 μg / kg、91μg / kg、90μg / kg、89μg / kg、88μg / kg、87μg / kg、86μg / kg、85μg / kg、84μg / kg、83μg / kg、82μg / kg、81μg / kg、80μg / kg、79μg / kg、78μg / kg、77μg / kg g、76μg / kg、75μg / kg、74μg / kg、73μg / kg、72μg / kg、71μg / kg、70μg / kg、69μg / kg、68μg / kg、67μg / kg、66μg / kg、65μg / kg、64μg / kg、63μg / kg、62μg / kg、61μg / kg g / kg、60μg / kg、59μg / kg、58μg / kg、57μg / kg、56μg / kg、55μg / kg、54μg / kg、53μg / kg、52μg / kg、51μg / kg、50μg / kg、49μg / kg、48μg / kg、47μg / kg、46μg / kg 、45μg / kg、44μg / kg、43μg / kg、42μg / kg、41μg / kg、40μg / kg、39μg / kg、38μg / kg、37μg / kg、36μg / kg、35μg / kg、34μg / kg、33μg / kg、32μg / kg、31μg / kg、30μg / kg g / kg、29μg / kg、28μg / kg、27μg / kg、26μg / kg、25μg / kg、24μg / kg、23μg / kg、22μg / kg、21μg / kg、20μg / kg、19μg / kg、18μg / kg、17μg / kg、16μg / kg、15μg / kg 、14μg / kg、13μg / kg、12μg / kg、11μg / kg、10μg / kg、9μg / kg、8μg / kg、7μg / kg、6μg / kg、5μg / kg、4μg / kg、3μg / kg、2μg / kg、1μg / kg as well as all the ranges formed by these values as limit values。For example, the methyl mercaptan content will be from 100 μg / kg to 50 μg / kg; preferentially from 90 μg / kg to 60 μg / kg, preferentially from 80 μg / kg to 60 μg / kg, even more preferentially from 70 μg / kg to 60 μg / kg.

[0150] The vegetable protein isolate according to the invention is preferably characterized in that its degree of hydrolysis (or DH) is less than 10%, preferentially less than 5.

[0151] Likewise, the invention relates to the use of the plant protein isolate obtained using the strain CNCM I-5802 in nutritional preparations in dairy or vegetable drinks, in yogurt-type fermented milks (blended, Greek-style, drinkable fermented milks), and in dairy or vegetable creams, frozen desserts or sorbets.

[0152] Finally, the present invention relates to the use of the isolate according to the invention in 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 small cereals ("crisps") obtained by cooking-extrusion, wherein more particularly a high-protein solution is sought that has no negative impact on the preparation process or on the texture of the final preparation or final product.

[0153] 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.

[0154] 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.

[0155] All percentages, parts and ratios used herein are by weight of the total formulation, unless otherwise specified.

[0156] 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.

[0157] 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.

[0158] 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.

[0159] In an exemplary embodiment, the powdered nutritional formulation may be formulated for:

[0160] - Repair muscles after intense effort, such as in athletes,

[0161] -Maintain or build muscle mass in athletes, or

[0162] - As a meal replacement for people trying to lose weight through its satiogenic effect.

[0163] 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.

[0164] Powdered food formulations can have a protein content suitable for the nutritional needs of the end user, but in most cases the reconstituted powder contains 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.

[0165] 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.

[0166] For example, the legume protein isolate according to the present invention (preferably pea or bean protein isolate) can comprise 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, or 90%-100% of the total protein content of the preparation, or any combination of these percentage ranges. 100% is the final preferred embodiment for maximizing the effect of FGF19 overexpression.

[0167] 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.

[0168] 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.

[0169] 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.

[0170] In particular, the nutritional preparations according to the invention can be used in the following areas:

[0171] -Diet and nutrition (exercise, weight loss),

[0172] - clinical nutrition (in the form of a drink, dessert cream or enteral bag),

[0173] - Dairy products (in the form of yogurt, dairy drinks, dairy cream, frozen desserts or sorbets).

[0174] -Biscuit products, pastry products, bread-making products and high-protein cereal products.

[0175] 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.

[0176] 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.

[0177] Ready-to-drink protein-enriched or high-protein beverages thus provide the body with an intake of selected proteins with fewer calories.

[0178] These high-protein drinks must:

[0179] -Rich in protein, low in carbohydrates and fat;

[0180] -Has a good taste;

[0181] - Designed to aid weight loss, stimulate fat burning and aid muscle recovery;

[0182] - is full;

[0183] -Can be used to manage cravings without any added sugar or fat;

[0184] -Has a balanced content of essential amino acids, fiber, vitamins and minerals;

[0185] -Low calories.

[0186] These ready-to-drink beverages can advantageously be prepared with the legume 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.

[0187] 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).

[0188] 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%.

[0189] 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.

[0190] 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.

[0191] 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.

[0192] Diet drinks are known to be ideal for aiding weight loss. More specifically, they make it possible to:

[0193] - Provides a satiety effect

[0194] - Protects muscles and tones the body to avoid weight gain.

[0195] Like "sports" drinks, these diet drinks have:

[0196] -Balanced content of essential amino acids, fiber, vitamins and minerals

[0197] - Low in sugar, fat and calories.

[0198] 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).

[0199] 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.

[0200] 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.

[0201] These formulations are typically packaged in flexible bags (made of PVC) and administered through a nasogastric tube, gastrostomy, nasojejunal tube, nasoduodenal tube, or jejunostomy.

[0202] These nutrient blends consist of proteins, lipids, carbohydrates, vitamins, and minerals, with or without fiber.

[0203] 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.):

[0204] -Polymer mixture

[0205] - Low calorie (0.5kcal / ml-0.75kcal / ml), normal or high protein, with or without fiber

[0206] - Isocaloric (1kcal / ml), normal or high protein, with or without fiber

[0207] - High calorie (1.25kcal / mL-1.5kcal / mL), normal or high protein, with or without fiber

[0208] -Specific formulations (disturbance of blood sugar metabolism, respiratory failure).

[0209] Semi-elemental blends are isocaloric or hypercaloric, normal or high protein blends based on medium chain peptides and triglycerides.

[0210] Pea protein isolate as a protein source is particularly suitable for this purpose due to its functional properties.

[0211] Furthermore, they make it possible to maintain the same properties as milk proteins at a lower cost.

[0212] 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.

[0213] Example

[0214] Example 1: Method according to the invention starting from pea flour

[0215] 2 kg (in commercial weight) of yellow smooth pea flour (85% dry matter and 28% protein N6.25, calculated on a dry weight basis) was diluted with deionized water (20°C). The resulting suspension was homogenized for 30 minutes using a Rayneri table mixer equipped with a dispersing turbine. The resulting suspension was centrifuged at 1000 x g for 5 minutes in a Beckman centrifuge. After removing the pellet containing the insoluble fraction (primarily a fiber / starch mixture), approximately 7 kg of supernatant with a dry matter content of 7% (DM) was recovered. The resulting supernatant was designated "crude extract" and standardized by adjusting its dry matter content to 6%.

[0216] This set of steps leading to a crude extract is hereinafter referred to as the "process head-end".

[0217] The crude extract was then acidified according to the following protocol:

[0218] Pre-culture

[0219] -Strain: CNCM I-5802

[0220] -Inoculum: 1 frozen bead

[0221] - Culture medium: MRS broth (Man-Rogosa-Sharpe), ready to use, Sigma ref. 69966

[0222] - Incubate at 37°C for 24 hours

[0223] - The obtained biomass was concentrated and washed 3 times with physiological water at 4°C

[0224] - Based on the method described above for preparing inoculum A, calculate the amount to be added to 900 mL of crude extract as 6% DM in order to obtain 10 9 The cell concentration of CFU / mL (OD = 1 <->

[0225] 3×10 8 CFU / mL)

[0226] nourish :

[0227] -2L Fermentation tank.

[0228] -Reaction volume: 900 mL crude extract at 4°C

[0229] - Stirring was controlled at 300 rpm and the temperature was 40°C

[0230] - Adjust pH to 7 before inoculation to normalize

[0231] - inoculating the fermenter with an amount of preculture that allows obtaining 10 9 The cell concentration is expressed as CFU / mL.

[0232] -use pH meter to monitor changes in pH (natural acidification of the extract by the strain)

[0233] - Run parallel controls under the same conditions, but without the introduction of the strain, in order to monitor the absence of harmful contaminants that could compromise the method

[0234] The acidification reaction parameters are as follows:

[0235] -Control:

[0236] -Control maintained at pH 7, without acidification, to verify the absence of contaminants

[0237] - in the presence of strains:

[0238] - Acidification time to pH 5 is 110 minutes

[0239] -Vm (or average acidification rate) is 2 pH units of acidification in 110 minutes, or -0.018 pH units / minute (expressed as pH units / minute, with the '-' symbol indicating acidification)

[0240] -VM (or Maximum Acidification Rate) was -0.027 pH units / min, recorded at pH 6.24 after 42 minutes of reaction.

[0241] It is noted that the fermentation times are very short compared to the teaching of Emkani et al., 2021 (Emkani et al., Foods, 10, 549, 2021), which describes fermentation times between 350 and 500 minutes depending on the strain, ie more than 3 times that of strain CNCM 1-5802.

[0242] At the end of the acidification, the suspension was centrifuged at 1000 x g for 5 minutes at 6°C. The supernatant was removed (which could be retained for analysis) and replaced with the same amount of sterile deionized water. The resulting suspension was stirred to homogenize it and the pH was adjusted to 7. After stirring for 15 minutes and visually inspecting the homogeneity, the suspension was lyophilized for analysis with reference to "Example 1 according to the present invention."

[0243] Example 2: Repeatability of Example 1 :

[0244] The principle here is to use identical pea flour to repeat identical method described in embodiment 1 for three times.

[0245] The parameters of the acidification reaction are summarized in Table 1 below:

[0246] [Table 1]

[0247]

[0248]

[0249] At the end of the acidification, the suspension was centrifuged at 1000 x g for 5 minutes at 6°C. The supernatant was removed (which could be retained for analysis) and replaced with the same amount of sterile deionized water. The resulting suspension was stirred to homogenize it and the pH was adjusted to 7. After stirring for 15 minutes and visually inspecting the homogeneity, the suspension was lyophilized and analyzed with reference to "Examples 2.1 / 2.2 / 2.3 According to the Invention."

[0250] Example 3: Process according to the invention starting from pea isolate

[0251] The method can be applied to pea protein isolate (e.g. from Roquette F85M), making it possible to simplify the front end of the process of Example 1. A suspension with 6% dry matter was produced using only pea protein isolate and drinking water.

[0252] The suspension was then acidified according to the following protocol:

[0253] Pre-culture

[0254] -Strain: CNCM I-5802

[0255] -Inoculum: 1 frozen bead

[0256] - Culture medium: MRS broth (Man-Rogosa-Sharpe), ready to use, Sigma ref. 69966

[0257] - Incubate at 37°C for 24 hours

[0258] - The obtained biomass was concentrated and washed 3 times with physiological water at 4°C

[0259] - According to the method described above for preparing inoculum A, the amount to be added to 900 ml of crude extract was calculated to be 6% in order to obtain 10 9 The cell concentration of CFU / mL (OD = 1 <-> 3 × 10p 8

[0260] CFU / mL)

[0261] nourish :

[0262] -2L Fermentation tank.

[0263] -Reaction volume: 900 mL crude extract at 4°C

[0264] - Stirring was controlled at 300 rpm and the temperature was 40°C

[0265] - The pH was adjusted to 7 without manipulation (natural acidification of the strain).

[0266] -use pH meter to monitor pH changes

[0267] - Run parallel controls under the same conditions, but without the introduction of the strain, in order to monitor the absence of harmful contaminants that could compromise the method

[0268] Example 4: Process not according to the invention - acidification with hydrochloric acid:

[0269] The process was performed so as to produce protein flocs representative of those obtained by conventional methods of the prior art.

[0270] The front end of the process was the same as in Example 1 in order to produce a crude extract with 6% DM.

[0271] Acidify with stirring and adjust the pH to 5 with 1N HCl. Apply a temperature of 60° C. and maintain for 10 minutes.

[0272] At the end of the acidification, the suspension was centrifuged at 1000 × g for 5 minutes at 6°C. The supernatant was removed (which could be retained for analysis) and replaced with the same amount of sterile deionized water. The resulting suspension was stirred to homogenize it and the pH was adjusted to 7. After stirring for 15 minutes and visually inspecting the homogeneity, the suspension was lyophilized for analysis with reference to "Example 4-HCl acidification not according to the invention"

[0273] Example 5: Method not according to the invention - acidification with strains not possible to carry out and / or obtain the invention :

[0274] The purpose here is to demonstrate the importance of the strain used. The protocol is almost identical to that described in Example 1.

[0275] 2 kg (in commercial weight) of yellow smooth pea flour (85% dry matter and 28% protein N6.25, calculated on a dry weight basis) was diluted with deionized water (20°C). The resulting suspension was homogenized for 30 minutes using a Rayneri table mixer equipped with a dispersing turbine. The resulting suspension was centrifuged at 1000 x g for 5 minutes in a Beckman centrifuge. After removing the pellet containing the insoluble fraction (mainly a fiber / starch mixture), approximately 7 kg of supernatant with 7% substance was recovered. The resulting supernatant was designated "crude extract" and standardized by adjusting its dry matter to 6%.

[0276] The crude extract was then acidified according to the following protocol:

[0277] Pre-culture

[0278] - Strains: Compare several strains. The list will be presented in the table below

[0279] -Inoculum: 1 frozen bead

[0280] - Culture medium: MRS broth (Man-Rogosa-Sharpe), ready to use, Sigma ref. 69966

[0281] - Incubate at 37°C for 24 hours

[0282] - The obtained biomass was concentrated and washed 3 times with physiological water at 4°C

[0283] - According to the method described above for preparing inoculum A, the amount to be added to 900 ml of crude extract was calculated to be 6% in order to obtain 10 9 CFU / mL cell concentration (OD = 1 <-> 3 × 10p8

[0284] CFU / mL)

[0285] nourish :

[0286] -2L Fermentation tank.

[0287] -Reaction volume: 900 mL crude extract at 4°C

[0288] - Stirring was controlled at 300 rpm and the temperature was 40°C

[0289] - The pH was adjusted to 7 without manipulation (natural acidification of the strain).

[0290] -use pH meter to monitor pH changes

[0291] - Parallel controls are performed under the same conditions, but without the introduction of the strain, in order to monitor the absence of harmful contaminants that could disrupt the method.

[0292] The acidification reaction parameters are as follows:

[0293] [Table 2]

[0294]

[0295] It is clear that only the strain recommended for carrying out the present invention (in bold) makes it possible to achieve acidification to pH 5 in less than 130 minutes. This is an undeniable advantage for an industrial process.

[0296] For a comprehensive evaluation, several analyses were performed, including analysis of organic volatile compounds (including methyl mercaptan) by GPC / MS, and measurement of the degree of hydrolysis.

[0297] A mass balance (in bold) was also performed for the recovery of protein flocs obtained using the strains recommended for this invention. This was calculated from 1) the amount of dry matter used in the crude extract and 2) the amount of dry matter obtained in the flocs. The ratio of the two (2 / 1) gives the extraction yield. This was compared with Example 4 (acidified with HCl). The results are summarized in Table 3 below:

[0298] [Table 3]

[0299] Yield (%, sd = + / - 0.02) Example 4 - (not according to the invention - HCl) 56 Example 1 (the present invention) 63 Enterococci 61 Lactobacillus delbrueckii subsp. jacobsenii 66 Lactobacillus mucosa 63 Lactobacillus salivarius 62 Streptococcus salivarius 62

[0300] Sd represents standard deviation.

[0301] Example 6: Industrial pilot scale verification of the present invention

[0302] To facilitate the execution of these tests, and to ensure a strict comparison of the conditions, the selected feedstocks were produced using the same procedure as for the front end of the process described in Example 1 and then atomized to stabilize them.

[0303] Table 4: Physicochemical characteristics of atomized crude extracts [Table 4]

[0304]

[0305]

[0306] Four comparative tests and their associated prototypes are described in Table 5. They were performed according to the following procedure: - Rehydration of the atomized crude extract with 6% DM

[0307] - Acidification in 20L Biolaffite fermenter:

[0308] For the three "fermentation" tests, the reaction method was that described in paragraph 130, but applied to a 20-liter volume.

[0309] For the "HCl" control, HCl was introduced to adjust the pH to 5

[0310] - Various acidifications are followed by heating steps aimed at precipitating any proteins that may still be in solution:

[0311] Continuous: Tubular exchange zone with the following parameters: preheat to 35°C, hold at 74°C for 10 seconds, cool to 65°C

[0312] Batch: Heating and stirring of the reaction vessel under control - 60°C - 30 minutes - Work-up of the fermentation broth (must):

[0313] 4000×G centrifugation

[0314] .Recovery of sediment

[0315] Redisperse with water to obtain 15% DM suspension

[0316] Adjust to pH 7

[0317] HTST treatment (preheating to 50°C / heating to 130°C for 2 seconds / rapid cooling to 75°C)

[0318] .Atomization (T℃ air inlet = 200℃ / T℃ air outlet = 90℃)

[0319] Table 5 below summarizes the different tests:

[0320] [Table 5]

[0321]

[0322]

[0323] The concept here is to verify that the process according to the invention works on a 20 liter pilot scale and to verify the effect of combining with heating to increase precipitation yield.

[0324] Biochemical and physicochemical analyses were performed and are summarized in Table 6 below:

[0325] [Table 6]

[0326]

[0327] Interestingly, in contrast to the article by Emkani et al., 2021 (Emkani et al., Foods, 10, 549, 2021), the degree of hydrolysis remains unchanged when the method according to the present invention is carried out. This is in stark contrast to Emkani et al., 2021 (Emkani et al., Foods, 10, 549, 2021), as there is no hydrolysis and, from this perspective, the product remains intact and unchanged. This is particularly important when an additional extrusion step is performed. In practice, extrusion performed on hydrolyzed proteins is less effective.

[0328] Example 5: Characterization of the organic volatile composition of the generated samples :

[0329] Several samples produced above were subjected to GPC / MS analysis in order to determine the various organic volatile compounds.

[0330] Comparison was made between "Sample 1" (according to the invention) and "Sample 4" (prior art control - HCl acidification). The results are presented in Table 7:

[0331] [Table 7]

[0332]

[0333]

[0334] It is readily apparent that the process according to the present invention reduces all organic volatile compounds by at least 75% compared to the prior art, with the exception of methyl acetate (the level of which remains unchanged) and ethyl acetate (the concentration of which is tripled). Specifically, the methyl mercaptan content is reduced (approximately 15% of the prior art value). Methyl mercaptan is a compound that, at low concentrations (ppb range), produces a sulfur-like flavor in foods such as ready-to-drink beverages or wet protein extrudates.

[0335] Use the following protocol to accurately quantify methyl mercaptan:

[0336] After the addition of internal standards (hexanal-d12 and dimethyl sulfide-d6), an aliquot of each sample was diluted in water and subjected to solid phase microextraction (SPME). Desorption was performed in the chromatographic injector.

[0337] -Analysis was performed by gas chromatography coupled with mass spectrometry (GC-MS) under the following operating conditions:

[0338] Chromatograph: Shimadzu 2010

[0339] ○Chromatographic column: PDMS

[0340] ○Injection: No split / Split

[0341] ○Mass spectrometer: Shimadzu QP2010+

[0342] ○Ionization method: electron bombardment (70eV)

[0343] ○Detection mode: Scan

[0344] - Identification and quantification of hexanal and methyl mercaptan were based on 1-point calibration with the same reference compounds.

[0345] The results obtained are as follows:

[0346]

[0347] It was observed that by practicing the present invention, the hexanal content was half that of the prior art.

[0348] Methyl mercaptan was also less than 100 μg / kg, which is much higher when acid precipitation is performed as in the prior art.

[0349]

Claims

1. A method for extracting plant protein, comprising the following steps:

1. suspending the plant powder in a preferred aqueous solvent to obtain a plant powder suspension; 2a. Adding a strain to the plant powder suspension of step 1 to form an inoculated suspension, the strain being selected from the group consisting of: Lactobacillus fermentum, Lactobacillus salivarius, Lactobacillus delbrueckii subsp. Jacobsenii, Lactobacillus mucosae, Streptococcus salivarius and Enterococcus lactis; the Lactobacillus strain being preferably a Lactobacillus fermentum strain; even more preferably a strain deposited at CNCM under the number CNCM I-5802; 2b. incubating the inoculated suspension of step 2a so that the strain acidifies the inoculated suspension until a protein precipitate is formed; 3. Isolation of the protein precipitate obtained at the end of step 2b.

2. The method according to claim 1, characterized in that The plant meal of step 1 is in the form of plant powder, which contains protein in an amount greater than 1 wt%, preferably 15 wt% to 30 wt%, relative to the weight of the powder.

3. The method according to claim 1 , wherein The vegetable meal of step 1 is an isolate or a concentrate, preferably an isolate having a protein content greater than 80% by dry weight relative to the dry matter of the isolate.

4. The method according to any one of claims 1 to 3, characterized in that The plant powder of step 1 is a leguminous plant powder, preferably characterized in that the plant powder is a leguminous plant powder selected from the group consisting of peas and broad beans, more preferably characterized in that the plant powder is pea powder.

5. The method according to any one of claims 1 to 4, characterized in that The incubation temperature of the suspension in step 2b is 30°C to 50°C, preferably 35°C to 45°C.

6. The method according to any one of claims 1 to 5, characterized in that Before carrying out step 2a, the pH of the plant powder suspension of step 1 is adjusted to 6.5 to 7.5, preferably to 7.

0.

7. The method according to any one of claims 1 to 6, characterized in that The strains described in step 2a were added to obtain a concentration of 0.1×10 Lactobacillus per ml of plant powder suspension. 9 to 1×10 9 The suspension was inoculated with a colony forming unit (CFU).

8. The method according to any one of claims 1 to 7, characterized in that The incubation of step 2b is performed until the inoculated suspension has a pH of 4 to 5, preferably a pH of 5.

0.

9. The method according to any one of claims 1 to 8, characterized in that If at the end of step 2b the pH of the seeded suspension is not between 4 and 5, step 2b comprises at the end of step 2b a sub-step 2b' of acidifying the seeded suspension by adding acid so that the seeded suspension obtained at the end of step 2b has a pH between 4 and 5, preferably a pH between 4.5 and 5.

10. The method according to any one of claims 1 to 9, characterized in that Step 2b comprises, at the end of step 2b, a sub-step 2b'' of additional heating in order to increase the flocculation yield, said sub-step 2b'' consisting in bringing the plant powder suspension of step 2b to a temperature of 45°C to 85°C.

11. The method according to any one of claims 1 to 10, characterized in that Step 3 includes the following steps: 3a. The pH of the inoculated suspension obtained at the end of step 2b is adjusted to a pH of 6 to 9, preferably a pH of 7, preferably by adding soda or lime to the suspension; 3b. heat treating the suspension of step 3a at 100°C to 160°C for 0.1 to 1 second, and 3c. drying the plant powder suspension of step 3b by means of a sprayer until a plant powder isolate having a dry matter greater than 95% is obtained.

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

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

14. The plant protein isolate according to any one of claims 12 or 13, characterized in that The methyl mercaptan content is reduced by more than 25%, preferably more than 50%, relative to an isolate obtained by acidifying the plant powder suspension by adding hydrochloric acid alone.

15. The plant protein isolate according to any one of claims 12 to 14, characterized in that The protein content of the isolate is between 80% and 95% by weight, preferably between 82% and 92% by weight, preferably between 84% and 90% by weight, preferably between 84% and 88% by weight, relative to the total weight of the dry matter of the isolate.

16. A plant protein isolate, characterized in that Its hexanal content is less than 6000 μg / kg and its methyl mercaptan content is less than 100 μg / kg.

17. The plant protein isolate according to claim 16, characterized in that The hexanal content is less than 5500 μg / kg, preferably less than 5000 μg / kg, preferably less than 4500 μg / kg, preferably 4000 μg / kg, preferably 3500 μg / kg.

18. The plant protein isolate according to claim 16 or 17, characterized in that The methyl mercaptan content is less than 120 μg / kg, preferably less than 110 μg / kg, preferably less than 100 μg / kg, preferably less than 90 μg / kg, preferably 80 μg / kg, preferably 70 μg / kg, and preferably 65 μg / kg.

19. Use of an isolate according to any one of claims 12 to 18 or an isolate obtained according to any one of claims 1 to 11 for industrial applications including food, nutraceutical and pharmaceutical applications.

20. A microbial strain deposited at the CNCM under number CNCM 1-5802.

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