Improved pea or broad bean proteins

Abstract

The present invention relates to pea proteins having milk flavor characteristics, to a method for producing said pea proteins and to the use of said proteins for the production of food or beverage products, in particular plant-based milk substitutes.

Description

Technical Field

[0001] The present invention relates to a novel composition comprising pea or faba bean protein associated with soluble fiber. Another object of the invention relates to a process for producing these novel compositions of pea protein. The invention also relates to the use of said compositions of pea or faba bean protein in the production of food products. Existing technology

[0002] Daily protein requirements typically range from 12% to 20% of food intake, with these being provided equally by products of animal origin (meat, fish, eggs, dairy products) and by plant foods (cereals, legumes, seaweed).

[0003] In developed countries, protein intake is still mainly in the form of proteins of animal origin. These proteins have good nutritional properties and interesting functional properties, which make them suitable for use in a wide variety of food products.

[0004] However, numerous studies have shown that excessive consumption of protein from animal sources to the detriment of plant proteins is one of the reasons for the increase in cancer and cardiovascular diseases. In addition, animal proteins have many disadvantages in terms of their allergenicity (especially proteins from milk or eggs) and in terms of the environment associated with the harmful effects of intensive farming.

[0005] Consequently, manufacturers are increasingly demanding plant-based proteins that possess beneficial nutritional and functional properties without the drawbacks of animal-based proteins.

[0006] Since the 1970s, pea has been the most exploited legume in Europe (primarily in France), particularly as a protein source intended for animal and human food. 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. Pea proteins (primarily vicilin) ​​have been extracted and utilized industrially for many years.

[0007] The field bean, also known as the faba bean, is a plant of the species Vicia faba. It is a legume in the family Fabaceae, subfamily Faboideae, and tribe Fabeae. It is the same species as the broad bean, a plant that has been used for human consumption since ancient times. Therefore, the word bean refers to both the seed and the plant.

[0008] First, for pea or fava bean protein, there are so-called "dry extraction" methods. The principle of these methods is to grind the seeds into a powder, which is then introduced into a turboseparator, a device used to sort the particles according to their size and density in an air stream. Turbine separation produces a protein-rich fraction and a starch-rich fraction. As will be explained later, the protein-rich fraction is approximately 40%-60% and still contains between 2% and 15% starch. This starch is a polysaccharide fraction, which is not necessarily desirable because it contributes to raising blood sugar levels. It would be interesting to replace it with polysaccharides that are not digestible by the consumer but can be digested by its digestive microflora. These same properties are also observed in the case of fava beans.

[0009] Another example of a pea or broad bean protein extraction method is patent EP1400537, which is a typical so-called "wet" extraction method. In this method, the seeds are ground in the absence of water (a method called "dry grinding") to obtain a powder. This powder is then suspended in water at room temperature and the various steps of protein extraction are then carried out. This type of method separates proteins belonging to the globulin subgroup (about 80% of pea proteins) and proteins belonging to the albumin subgroup (about 20% of pea proteins) by isoelectric precipitation. After recovering the flocs composed mainly of globulins, these remain in the liquid fraction. These albumins are in solution together with galacto-oligosaccharides (GOS) from peas that are not easily digested by humans, salts (mainly potassium, which can be harmful in high doses) and other anti-nutritional factors (such as antitrypsin factors). These same characteristics are also observed in the case of broad beans.

[0010] Several problems are readily apparent, such as the separation of proteins into two fractions, which necessitates the evaluation of two different fractions, or the combination of GOS with albumin, which is difficult to digest and requires post-processing, making the process more complex and expensive. Finally, the high presence of starch in the protein-rich fraction of the concentrate obtained by dry turbine separation is also a nutritional disadvantage for certain preparations.

[0011] After extensive research, the Applicant has therefore developed a new production process which makes it possible to provide a composition containing all the so-called soluble proteins of pea or bean, ie including all the globulins and albumins, and a certain amount of soluble fiber derived from GOS, making it possible to obtain a high-quality nutritional source. Summary of the Invention

[0012] Therefore, the present invention relates to a method for producing pea or broad bean protein, comprising the following steps:

[0013] 1. preparing an aqueous suspension of ground pea or bean seeds in an aqueous solution, optionally in the presence of a heat treatment;

[0014] 2. removing the insoluble fraction by solid / liquid separation of the aqueous suspension of ground pea or bean seeds obtained in step 1) to obtain a protein-rich fraction;

[0015] 3. Defructosylation of galacto-oligosaccharides from the protein-rich fraction by enzymatic and / or fermentative means.

[0016] Preferably, the aqueous suspension of ground pea or bean seeds is obtained by adding pea or bean flour obtained by dry milling before dispersing the pea or bean flour in the aqueous solution.

[0017] When the pea or bean seeds are introduced into the aqueous solution as whole pea or bean seeds, step 1 of the method comprises a step of wet-grinding the aqueous composition comprising the pea or bean seeds so as to obtain an aqueous suspension of ground pea or bean seeds.

[0018] In the absence of the optional heat treatment in step 1, the aqueous solution and the resulting aqueous suspension are at a temperature between 5° C. and 30° C., preferably at ambient temperature. Ambient temperature is understood to mean a temperature between 15° C. and 27° C., preferentially between 19° C. and 23° C., wherein the temperature values ​​may be 15° C., 16° C., 17° C., 18° C., 19° C., 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C. or 27° C.

[0019] Preferably, the optional heat treatment of step 1 comprises:

[0020] a) introducing pea or bean seeds or ground pea or bean seeds into an aqueous solution at a temperature between 65° C. and 90° C. so as to obtain an aqueous composition comprising ground pea or bean seeds;

[0021] b) heat-treating the aqueous composition obtained in step a) at a temperature between 40° C. and 65° C. for 1 to 10 minutes.

[0022] Preferably, the defructosylation of galacto-oligosaccharides (GOS) in step 3 is performed using an enzyme selected from the group consisting of invertase, α-galactosidase, β-fructosidase and any combination thereof.

[0023] Alternatively, the defructosylation of galacto-oligosaccharides (GOS) is carried out using a microorganism of the genus Bacillus, preferentially Bacillus subtilis, preferentially a strain of Bacillus subtilis, as deposited with the CNCM on May 28, 2020 under number I-5515.

[0024] Another object of the invention relates to pea or faba bean protein, characterized in that it comprises between 50% and 70% protein content, expressed in grams of protein per 100 g of dry matter, said protein consisting of a mixture of globulins and albumins; and between 3% and 15% soluble fibers derived from pea galacto-oligosaccharides.

[0025] Preferably, the globulin / albumin dry weight ratio is between 70:30 and 90:10, preferentially between 75:25 and 85:15.

[0026] Preferably, the soluble fiber derived from pea or bean galacto-oligosaccharides is selected from the list comprising melibiose, mannotriose, verbascotetraose and mixtures thereof.

[0027] Another object of the present invention also relates to the use of said composition of pea or bean protein according to the invention for producing a food or beverage product, in particular a plant-based milk substitute. DETAILED DESCRIPTION

[0028] The present invention relates to a method for producing pea or broad bean protein, comprising the following steps:

[0029] 1. preparing an aqueous suspension of ground pea or bean seeds in an aqueous solution, optionally in the presence of a heat treatment;

[0030] 2. removing the insoluble fraction by solid / liquid separation of the aqueous suspension of ground pea or bean seeds in step 1) to obtain a protein fraction;

[0031] 3. Defructosylation of galacto-oligosaccharides from the protein fraction by enzymatic and / or fermentative means.

[0032] Step 1) involves introducing peas or broad beans into an aqueous solution. The pea or broad bean dumplings used in step 1) may have previously undergone steps well known to those skilled in the art, such as, in particular, cleaning (removal of unwanted particles such as stones, dead insects, soil residues, etc.) or even removal of the outer fibers (outer cellulose shell) of the peas or broad beans by a well-known step known as "shelling".

[0033] Therefore, the term "peas" or "beans" in step 1) should be understood to mean whole peas or beans or peas or beans in the form of cotyledons, the outer skin of which has been preferably removed. Alternatively, ground pea seeds (i.e. pea flour) can be used, these ground peas usually being obtained by dry milling.

[0034] According to the general knowledge of those skilled in the art, the seeds are ground. In fact, those skilled in the art will first know how to select a suitable mill, mainly selected from the group of grinding mills or impact mills. According to the quality of the selected seeds (for example, its residual moisture content), the technician will be able to adjust the parameters of the mill to achieve the required powder quality. The particle size is determined by the technician considering its ultimate goal (i.e., wet extraction method). Preferably, the particle size is characterized by a D mode between 15 microns and 300 microns, preferentially between 10 microns and 100 microns, or even more preferentially between 20 microns and 50 microns. In order to measure this particle size, those skilled in the art will preferentially use a laser particle size analyzer. Even more preferably, the measurement of this particle size is to use a MALVERN 3000 laser particle size analyzer (equipped with a powder module) to carry out in the dry phase. The powder is placed in a feeder for the module, which has an opening in the range between 1mm and 4mm and a vibration frequency of 50% or 75%. The device automatically records the various sizes and adjusts the particle size distribution (or PSD) and the D-mode (D10, D50 and D90). The D-mode is well known to those skilled in the art and consists of the size of the largest particle population by number.

[0035] The aqueous solution may be water and may optionally contain additives such as, inter alia, antifoaming compounds, salts or bacteriostatic compounds.

[0036] The weight ratio of the amount of peas to the amount of aqueous solution in step 1) may in particular be between 0.1 and 2. In embodiments where the pea or bean seeds are ground into a flour before being introduced into the aqueous solution, the weight ratio of the amount of peas to the amount of aqueous solution in step 1) will be between 0.1 and 0.3, preferably between 0.15 and 0.25. Expressed as a percentage of dry matter, this ratio will be between 10% and 30%, preferably between 15% and 25%.

[0037] According to one variant, the pH of the suspension of step 1) is adjusted to between 8 and 10. This adjustment can be carried out by adding a base such as sodium hydroxide, lime or potash, preferably sodium hydroxide. According to another variant, the pH is not adjusted in this step.

[0038] In step 1) an aqueous suspension of ground pea or bean seeds is obtained by introducing pea or bean seeds or ground pea or bean seeds into an aqueous solution which, if a heat treatment is used, may optionally have been preheated to a temperature between 65° C. and 90° C.

[0039] In the case of a heat treatment, the temperature of the aqueous solution can be adjusted beforehand to a temperature of between 65°C and 90°C. The heating can be carried out using any device well known to those skilled in the art, such as an immersion heat exchanger. Preferably, the temperature is between 70°C and 80°C or even about 75°C.

[0040] Alternatively, the aqueous solution is not subjected to any preliminary heating and is directly heated to a temperature of between 65°C and 90°C when mixed with the peas or beans seeds or ground peas or beans seeds.

[0041] Preferably, the heat treatment temperature is between 40°C and 60°C, or even between 45°C and 55°C. Preferably, the heat treatment is carried out for 2 to 4 minutes.

[0042] According to any one of these embodiments, the method then comprises heat treating the suspension formed between the peas or beans seeds or ground peas or beans seeds and the aqueous solution to a temperature of between 40°C and 65°C for 1 to 10 minutes. The aqueous composition comprising the peas or beans seeds or ground peas or beans seeds can be heated or cooled to reach this temperature.

[0043] In the case where pea or bean seeds (and pea or bean flour that has not yet been ground) are used to prepare the suspension according to step 1), the method comprises a step of wet grinding an aqueous composition containing pea or bean seeds or ground pea or bean seeds in order to obtain an aqueous suspension of ground pea or bean seeds. If a heat treatment is applied, wet grinding is carried out after said heat treatment. Preferably, the method is carried out using pea or bean seeds and the wet grinding step is carried out by continuously passing through one or more mills to obtain an aqueous suspension of ground pea or bean seeds. The one or more mills can be any type of mill suitable for wet grinding, such as a wet ball mill, a wet cone mill, a wet spiral mill or a wet mill equipped with a rotor-stator system. According to a variant, the mill can be the mill used in the example of document WO2019 / 053387 in the name of the present applicant. In a variant in which the mill is of the rotor-stator type, this type of mill allows continuous grinding by passing the aqueous composition obtained between the pea or bean seeds and the aqueous solution through the mill. According to a preferred sub-variant, the method combines two cutting stages (pre-cutting and then cutting), using a different rotor-stator mill for each of these cuts. Pre-cutting and then cutting can be performed one after another, or alternatively, cutting can be performed after pre-cutting and then storing the aqueous composition obtained between the pea or bean seeds and the treated aqueous solution. Such a mill is disclosed in document WO2019 / 158589. Optionally, dilution with water can be performed during or at the end of this step to form an aqueous suspension of ground pea or bean seeds. According to a variant, water is added continuously or discontinuously during grinding to dilute the aqueous suspension. Generally speaking, the dry matter of the aqueous suspension of ground pea or bean seeds is in the range of 10% to 30%, for example 15% to 25%.

[0044] Step 2) of the method consists in extracting a component from the aqueous suspension of ground pea or lentil seeds and, in particular, in extracting a protein fraction by solid-liquid separation of a so-called insoluble fraction from the aqueous suspension of ground pea or lentil seeds. According to a variant, before the solid-liquid separation stage, a stage of adjusting the pH of the aqueous suspension of ground pea or lentil seeds can be performed. Thus, the solid-liquid separation can be performed after adjusting the pH of the aqueous suspension of ground pea or lentil seeds to a range of 6 to 9, preferably 8 to 9, most preferably 8.5 to 9. This pH adjustment stage can be performed in a stirred tank. This stage can be shorter or longer and lasts from 1 minute to 240 minutes, for example, typically from 5 minutes to 60 minutes. To perform the pH adjustment, any type of acid and / or base, organic or inorganic, or a mixture thereof can be added. Examples of acids that can be used include hydrochloric acid, sulfuric acid, citric acid or a mixture thereof. Examples of bases that can be used include sodium hydroxide, potassium base or lime and a mixture thereof. The addition of base or acid and the pH measurement can be performed on-line. The base and / or acid can be in the form of an aqueous solution. Advantageously, before this solid-liquid separation, the aqueous suspension of ground pea or lentil seeds is cooled to a temperature lower than 15°C. This temperature can in particular be in the range of 4°C to 14°C, for example 10°C to 12°C. This cooling step can be performed using known techniques such as, for example, passing the aqueous suspension of ground pea or lentil seeds through a heat exchanger.

[0045] Generally, the protein fraction is the soluble part of the aqueous suspension of ground pea or lentil seeds and the fraction rich in starch and fiber is the insoluble part. It is also possible to separate more than two insoluble fractions and, for example, to recover a first insoluble fraction rich in starch and a second insoluble fraction rich in fiber. Thus, according to a variant of the method, a fraction rich in starch and / or a fraction rich in fiber is recovered from the insoluble part resulting from the solid-liquid separation step 2).

[0046] The "fraction rich in starch and fraction rich in fiber" or "insoluble fraction" generally means a fraction comprising at least 50% of starch and / or fiber. Methods for quantifying starch and fiber are known to the person skilled in the art and specific methods are indicated later in the description. These fractions are generally recovered by known separation methods. The solid-liquid separation can in particular be performed by at least one separation step with a decanter, in particular a centrifugal decanter, a centrifuge or with a hydrocyclone. The method can likewise recover one or more fraction(s) rich in fiber and / or fraction(s) rich in starch removed from the suspension and recover the protein fraction useful for the remainder of the method of the application.

[0047] This step is key to distinguishing the concentrate obtained by the dry process from the concentrate obtained by the dry process. The protein-rich fraction obtained at the end of this step contains between 0% and 0.5% starch, unlike the concentrate obtained by turbine separation, which contains between 2% and 15%. This step will have a direct impact on the final composition obtained, as will be discussed later in this application.

[0048] At the end of step 2), the method may typically include adjusting the pH of the pea or faba bean protein to a pH range of 6 to 7.5, typically 6.5 to 7.5. This step may be performed by adding an inorganic or organic base (such as sodium hydroxide). The pH is typically increased by adding an alkaline aqueous solution.

[0049] Step 3)

[0050] The method then comprises the step 3) of defructosylation of the galacto-oligosaccharides by enzymatic and / or fermentative means.

[0051] For the purposes of the present invention, "galacto-oligosaccharide" refers to an oligomer formed by n sugars (monosaccharides) linked by α or β glycosidic bonds and naturally occurring in peas or beans, such as raffinose or stachyose.

[0052] Preferably, the water-soluble fraction according to the present invention comprises defructosylated galacto-oligosaccharides selected from the list comprising melibiose, mannotriose and verbascotetraose.

[0053] For the purposes of the present invention, the term "melibiose" means a disaccharide consisting of galactose units bonded to glucose units via α(1→6) glycosidic bonds.

[0054] For the purposes of the present invention, the term "mannotriose" means a trisaccharide consisting of a chain of one galactose unit bonded via an α(1→6) glycosidic bond to another galactose unit, which itself is bonded to a glucose unit via another α(1→6) bond.

[0055] For the purposes of the present invention, the term "verbosaccharide" (also called "mannotetraose") means a tetrasaccharide consisting of a chain of three galactose units bonded by an α(1→6) glycosidic bond, the third galactose unit itself being bonded to a glucose unit by another α(1→6) bond.

[0056] Any method known to those skilled in the art for quantifying these defructosylated galacto-oligosaccharides is suitable for the purposes of the present invention. Chromatographic methods are preferred. Preferably, those skilled in the art will use the HPAEC-PAD amperometric method, in particular using the following equipment:

[0057] -Dionex Carbopac PA1 4*50mm pre-column - Reference 43096

[0058] -Dionex Carbopac PA1 4*250-mm column - Reference 35391

[0059] - The detector is a PAD type, specifically a gold battery

[0060] - The eluent is:

[0061] □Solvent A / NaOH 0.1M: Stir 4 liters of water under helium (flow rate: 100 mL / min) for 15 minutes. Add 20 ml of 50% NaOH. Stir again under helium at 40 ml / min.

[0062] Solvent B / 0.1M NaOH + 0.5M Sodium Acetate: Weigh 82g of sodium acetate directly into a jar. Add 2l of H2O. Stir under helium (flow rate: 100ml / min) for 15 minutes, then add 10ml of 50% NaOH and stir again under helium. The helium flow rate can be reduced to 40ml / min.

[0063] Standards were used to calibrate the HPLC, and specifically:

[0064] Reagents Reference Melibiose Fluka reference 63630 Raffinose Sigma reference R-0514 Stachyose Sima reference S-4001 Vicianose Fluka reference 56217

[0065] An internal standard was also used: Panose reference SIGMA P-2407 60 mg in 100 ml water.

[0066] The injection volume was 5 μl at a temperature of 15° C. The analysis time was 90 minutes with a column temperature of 30° C. and an injector sensitivity of 300 nC or 5 μA.

[0067] The chromatographic elution conditions are as follows:

[0068]

[0069] The oxidation procedure of the PAD detector is as follows:

[0070] Time (min) Potential Integration 0 +0.05 0.20 +0.05 Start 0.40 +0.05 End 0.41 +0.75 0.60 +0.75 0.61 -0.15 1.0 -0.15

[0071] Calibration is performed by preparing a curve according to the following table:

[0072]

[0073] Take 1 ml of control (from 2 curves) + 1 ml of internal standard, and an appropriate amount of 20 ml of water.

[0074] Weigh the sample amount in mg, add 1 ml of internal standard and adjust to 20 ml of water.

[0075] Reference 4559T was filtered on GxF / GHP 0.45 pm.

[0076] In a first alternative, the defructosylation of galactooligosaccharides (GOS) is performed with an enzyme selected from invertases, a-galactosidases, b-fructosidases and any combination thereof.

[0077] Preferably, the enzyme used is an invertase, such as for example Sumizyme INV. The pH and temperature are adjusted in order to optimize the defructosylation reaction, such as for example for Sumizyme INV, pH 5 and 55°C. The reaction is monitored by performing an analysis, for example by HPLC analysis of the sugars present in solution. When the defructosylation is sufficient or even complete, it is neutralized with a base such as sodium hydroxide, then a heat treatment is performed in order to inhibit the enzyme, for example at 130°C for 10 seconds.

[0078] In a second alternative, the defructosylation of galactooligosaccharides (GOS) is performed with a microorganism of the genus Bacillus, preferentially Bacillus subtilis, preferentially Bacillus subtilis strain, as deposited on May 28, 2020 under the number I-5515 at CNCM.

[0079] For the purposes of the present invention, "fermentation" means a metabolic process that generally converts carbohydrates into acids, gases, or alcohols in order to extract some of their chemical energy while reoxidizing the coenzymes that are reduced by these reactions. It is a redox metabolic pathway in which the final electron acceptor is often confused with the final product of the reaction. It is characterized by partial degradation of fermentable substances and only allows limited energy production. It occurs in yeasts and bacteria, as well as in muscle cells under anaerobic conditions, i.e. under anaerobic conditions.

[0080] In this step of the method, it is important to defructosylate the GOS without modifying their carbon skeleton, so as not to hydrolyze them. In the background art, there are many solutions to solve this technical problem. The present invention preferably transforms them into fibers that are beneficial to human and animal nutrition.

[0081] Steps 1), 2), and 3) of the method are performed in this order. However, if deemed necessary by a skilled artisan, other optional steps, such as pH adjustment, may be implemented between steps 1), 2), and 3). Preferably, after the defructosylation step, the method may include a step 4) of additional heat treatment of the pea or faba bean protein. In this step, the temperature and time conditions may vary widely, for example, from 70°C to 140°C for a duration ranging from 0.1 seconds to several minutes. According to a first variant of this additional heat treatment step, the temperature range is 70°C to 90°C and the duration ranges from 0.1 seconds to 30 minutes. According to a second variant of this additional heat treatment step, the temperature range is 90°C to 110°C and the duration ranges from 0.1 seconds to 5 minutes. According to another variant, this additional heat treatment step is performed at a temperature of 110°C to 140°C for a duration of 0.1 seconds to 30 seconds, preferably 0.2 seconds to 15 seconds, for example 0.3 seconds to 10 seconds. The purpose of this step may be to functionalize and / or sterilize the pea protein. To perform this additional heat treatment step, the pea or faba bean protein can be in the form of an aqueous dispersion, preferably having a dry matter content ranging from 10% to 25%, for example, from 15% to 20%. Advantageously, the method of the present invention includes a step of cooling the pea protein after the additional heat treatment step. According to a preferred embodiment, this cooling step is achieved by rapid cooling. At the end of this step, the temperature can range from 60°C to 100°C, for example, between 70°C and 90°C. Similarly, the rapid cooling step is performed by applying a vacuum to the aqueous dispersion of pea protein, the vacuum applied being determined based on the selected cooling temperature.

[0082] According to one variant of the method, it includes an optional step 5) of shearing the pea or faba bean protein, for example by passing the aqueous dispersion of the protein through a high-pressure pump. As examples of high-pressure pumps, mention may be made of the high-pressure pumps sold by Silverson, also known as "high shear mixers," such as those of the UHS series. Preferably, the shearing step is performed by means of a high-pressure pump.

[0083] The shearing step may be performed before or after the heat treatment and / or pH raising steps.

[0084] According to another variant, the method additionally comprises an optional step 5) of homogenizing the pea proteins.

[0085] To perform this homogenization step, any type of homogenizer can be used. According to the application, it relates to an apparatus comprising a high-pressure pump and a homogenization head, wherein the apparatus is designed so that the product to be homogenized passes through the homogenization head under pressure. The homogenization head consists of a reduced-diameter opening, generally comprising a seat, a valve and a strike ring. Thus, passing the aqueous dispersion of pea or fava bean proteins through a homogenizer makes it possible to homogenize the pea proteins. The homogenization can be low-pressure homogenization, high-pressure homogenization or ultra-high-pressure homogenization. Depending on the homogenization technique used, the homogenization pressure can vary within a wide range of 1 bar to 1000 bar, for example 20 bar to 800 bar. According to one variant, the homogenization pressure ranges from 20 bar to 200 bar, for example from 50 bar to 150 bar. According to another variant, the homogenization pressure ranges from 200 bar to 800 bar, for example from 300 bar to 800 bar. According to one variant, the homogenization is single-action homogenization. According to another variant, the homogenization is multi-action homogenization, for example double-action homogenization. A homogenizer sold, for example, by GEA or Tetra Pak can be used.

[0086] The homogenization step can be performed before or after the heat treatment and / or the step of increasing the pH.

[0087] The method according to the application can also comprise an optional step 6) of membrane nanofiltration, preferably with a cut-off threshold of between 150 Da and 300 Da. This step makes it possible to pre-concentrate the composition before any potential subsequent evaporation and / or drying step and desalination. Tests have shown that, at a cut-off threshold of 300 Da, the ash content can be reduced from an initial 8% to 10% to a value of 5% to 7%.

[0088] To measure the ash content, the skilled person will use any method known in the art. Preferably, the skilled person will use the following method:

[0089] - Weigh a sample of peas P1

[0090] - Place the sample in an oven at 550°C for 24 hours

[0091] Weigh the new sample weight P2

[0092] Ash content = (P2 / P1)*100

[0093] The method according to the application can also comprise a drying step 7) of the pea or lentil proteins. Generally, this drying step is performed to reach a solid content greater than 80%, preferably greater than 90%, most preferably greater than 94% by weight of solids relative to the weight of the pea proteins. For this, any technique well known by the person skilled in the art can be used, such as freeze-drying, flash-drying or drum-drying, or atomization. The method can also comprise a step of grinding or micronization. Atomization is the preferred technique, in particular multi-effect atomization. The pea or lentil proteins can be in the form of a powder, the particle size d50 of which can vary greatly, for example from 10 pm to 500 pm, generally from 50 pm to 150 pm.

[0094] In the present application, "d50" means the particle size separating two populations by number, containing 50% and 50% respectively of the total amount of particles of the protein composition, measured in microns.

[0095] To perform this d50 measurement, a laser particle size analyzer is preferentially used, even more preferentially a Mastersizer 2000 from Malvern. The parameters used are as follows: liquid application, dispersed in ethanol; refractive index: 1.52; absorption index: 0.1 ; no ultrasound is used.

[0096] Preferentially, the drying step 7) can comprise a first sub-step of concentration by evaporation, followed by the actual drying step. This evaporation is generally performed using any suitable technique well known by the person skilled in the art, such as vacuum evaporation. The target dry matter content is between 15% and 30%, preferentially between 20% and 23%. This evaporation both eliminates undesirable volatile compounds and optimizes the drying step.

[0097] The steps 1) to 7) of the method can be performed in this precise order, but other optional steps, such as pH adjustment, can be implemented according to the needs of the skilled person.

[0098] Pea or fava bean proteins

[0099] Another object of the application relates to a composition of pea or lentil proteins that can be obtained by the method of the application.

[0100] Another object of the application relates to a composition of pea or lentil proteins, characterized in that it comprises a protein content, expressed in grams of protein per 100 g of dry matter, of between 50% and 70%, the proteins consisting of a mixture of globulins and albumins; and between 3% and 15% of soluble fibers derived from pea galacto-oligosaccharides.

[0101] "Composition of pea proteins" means a composition comprising mainly, but not exclusively, pea proteins. This composition can contain residual impurities, such as for example minerals, sugars, etc.

[0102] "Composition of faba bean protein" means a composition comprising mainly, but not exclusively, faba bean protein. Such a composition may contain residual impurities such as, for example, minerals, sugars, etc.

[0103] The term "pea" is to be understood in this application as including all wild varieties of "smooth pea" and all mutant varieties of "smooth pea" and "wrinkled pea". 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 mutant varieties of "smooth pea" and "wrinkled pea", regardless of the use (human food, animal feed and / or other uses) for which the varieties are typically used. The term "pea" in this application includes pea varieties belonging to the genus Pisum, and more specifically to sativum and aestivum. Such mutant varieties are in particular those known as "r mutants", "rb mutants", "rug 3 mutants", "rug 4 mutants", "rug 5 mutants" and "lam mutants" as described by CL HEYDLEY et al. in the article entitled "Developing novel pea starches", Proceedings of the Symposium of the Industrial Biochemistry and Biotechnology Group of the Biochemical Society, 1996, pp. 77-87.

[0104] The term "protein" is understood in the present application to mean a macromolecule formed by one or more polypeptide chains consisting of a series of amino acid residues linked to one another by peptide bonds. In the specific context of pea proteins, the present invention more specifically relates to globulins (approximately 50%-60% by weight of pea proteins) and albumins (20%-25% by weight of pea proteins).

[0105] For the purposes of the present invention, "globulin" means a protein that is soluble in neutral saline aqueous solution. Pea or bean globulins are mainly subdivided into three subfamilies: legumin, vicilin and convicilins.

[0106] For the purposes of this invention, the term "albumin" refers to proteins that are soluble in pure water. Pea albumin, present in about 20% of pea or faba bean protein, is mainly subdivided into two families, designated PA1 and PA2. Pea or faba bean albumin is mainly subdivided into two families, designated PA1 and PA2.

[0107] In general, the protein content of the pea or faba bean protein according to the present invention is between 50% and 70% by weight, expressed as grams of protein per 100g of dry matter. The protein content is the N6.25 content calculated using the Dumas method. Preferably, the protein content is between 50% and 70%, including 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69 or 70%.

[0108] The pea or bean protein according to the invention obviously usually contains other minor components besides protein, such as starch, lipids, fibers and / or sugars.

[0109] Generally speaking, the total starch content in the pea or bean protein produced according to the method of the present invention is between 0% and 0.5%, for example, 0.1% to 0.4%, and in particular 0.2% to 0.3%. This total starch content can be measured using the AOAC 996.11 method. Therefore, the residual starch content can be 0%, 0.1%, 0.2%, 0.3%, 0.4%, or 0.5%. This particularly low starch content is a characteristic of the pea or bean protein according to the present invention. The pea or bean protein concentrate obtained by turbine separation contains more starch, for example, between 2% and 5% according to the Feedipedia website (https: / / www.feedipedia.org / node / 7439), but more often up to 10% or more, as will be illustrated later in this application in the case of commercial concentrates. The presence of this residual starch has an impact not only on the nutritional characteristics of the product, but also on its functional properties, such as, for example, greater viscosity upon heating.

[0110] Generally, the total lipid content is 0% to 15%, such as 1% to 10%.The total lipid content can be determined by the AOAC 996.06 acid hydrolysis method.

[0111] Preferably, the globulin / albumin dry weight ratio is between 70:30 and 90:10, preferentially between 75:25 and 85:15.

[0112] Preferably, the pea or faba bean protein according to the invention has a degree of hydrolysis or DH of between 6% and 8%, preferentially between 6.5% and 7.5%. The DH value will therefore be 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%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9% or 8.0%.

[0113] For the purposes of the present invention, the expression "degree of hydrolysis" means the percentage between the amount of amine (or carboxyl) functional groups of free amino acids and the total amount, including free functional groups and those functional groups that participate in peptide bonds (chemical bonds characteristic of proteins, resulting from the association of the carboxyl functional group of a first amino acid with the amine functional group of a second amino acid). For a protein composition consisting of a chain of all its amino acids and therefore having only one free amine functional group and one free carboxyl functional group, the degree of hydrolysis would be 0%. Conversely, for a protein composition in which the same amino acids are all "free", i.e., their two amine and carboxyl functional groups are not involved in a peptide bond, the degree of hydrolysis would be 100%.

[0114] There are several methods for quantifying the degree of hydrolysis. They all consist primarily of a colorimetric determination of free amine (or carboxyl) functional groups, followed by a hydrolysis step aimed at breaking all peptide bonds, and finally a colorimetric determination of the total amine (or carboxyl) functional groups. The calculated percentage of free amine (or carboxyl) groups to total amine groups gives the degree of hydrolysis. Any known method can be used, such as the so-called TNBS method or the OPA method. In the present invention, the OPA method is preferred, and its measurement process is described as follows:

[0115] First, the amino nitrogen (free NH2) content of a protein sample according to the invention is determined using the MEGAZYME kit (reference number K-PANOPA). The protein nitrogen content (total nitrogen) of the sample is also determined. The degree of hydrolysis can then be calculated.

[0116] Determination of the content of amino nitrogen :

[0117] 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.

[0118] 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.

[0119] An accurately weighed sample P* of the sample to be analyzed is introduced into a 100 mL beaker. Based on the amino nitrogen content of the sample, the sample will be 0.5 g to 5.0 g. Approximately 50 ml of distilled water is added, homogenized and the mixture is poured into a 100 ml graduated flask. 5 ml of 20% sodium lauryl sulfate (SDS) is added and the mixture is supplemented with distilled water to a volume of 100 ml. 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.

[0120] Prepare blanks, standards, and samples directly in the spectrophotometer cuvettes under the following conditions:

[0121] -Blank: Introduce 3.00mL of solution 1 and 50μL of distilled water

[0122] - Standard: Introduce 3.00 mL of solution 1 and 50 μL of bottle 3 of the Megazyme kit

[0123] -Sample: Introduce 3.00 mL of Solution No. 1 and 50 μL of sample preparation.

[0124] The contents of each cuvette were mixed and absorbance measurements (A1) of the solutions were obtained 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).

[0125] 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.

[0126] The contents of each cuvette were mixed and then placed in the dark for approximately 20 minutes.

[0127] Then obtain absorbance measurements A2 of the blank, standards, and samples from the spectrophotometer at 340 nm.

[0128] The free amino nitrogen content, expressed as a weight percentage relative to the weight of the product, is given by the formula:

[0129]

[0130]

[0131] in:

[0132] ΔAech=Aech2–Aech1

[0133] ΔAblc=Ablc2–Ablc1

[0134] Aech2 = absorbance of the sample after adding solution 2

[0135] Aech1 = absorbance of the sample after adding solution 1

[0136] Ablc2 = absorbance of blank after adding solution 2

[0137] Ablc1=Absorbance of blank after adding solution 1

[0138] V = volume of the flask

[0139] m = weight of the sample (in g)

[0140] 6803 = Extinction coefficient of isoindole derivatives at 340 nm (in 1.mol -1 .cm -1 as units).

[0141] 14.01 = molar mass of nitrogen (in g.mol -1 (unit)

[0142] 3.15 = Final volume in the cuvette (in ml)

[0143] 0.05 = Sample in the cuvette (in ml)

[0144] Determination of the content of protein nitrogen :

[0145] 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.

[0146] Calculation of the degree of hydrolysis

[0147] The degree of hydrolysis (DH) was calculated using the following formula:

[0148]

[0149] The pea or faba bean protein according to the invention contains between 3% and 15% soluble fiber, preferably between 4% and 8% soluble fiber derived from pea galacto-oligosaccharides. Thus, the content may be between 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% and 15%.

[0150] Preferably, the soluble fiber derived from pea or bean galacto-oligosaccharides is selected from the list comprising melibiose, mannotriose, verbascotetraose and mixtures thereof.

[0151] The combination of proteins consisting of a mixture of globulins and albumins with defructosylated soluble fibers produces a unique ingredient of undeniable interest. With this single ingredient, it is possible to provide high-quality proteins and an amount of soluble fibers. In addition, according to the article “PREVENTION AND TREATMENT OF IRON DEFICIENCY ANEMIA IN WOMEN AND CHILDREN: IRON HOMEOSTASIS AND OPTIMIZING ABSORPTION USING PREBIOTICS AND BREAST MILK COMPONENTS” (Giorgetti, 2022), iron absorption is increased when consumed with GOS.

[0152] The characteristics of the pea or fava bean proteins can vary greatly depending on the parameters of the method previously described and as shown in the example section.

[0153] According to one embodiment, the solubility of the dried pea or fava bean protein at pH 7 ranges from 10% to 99%. The solubility can have all intermediate amounts (i.e., 11%, 12%, 13%... 97%, 98%, 99%) and the person skilled in the art will know, based on the method given above and in the example section, how to modify the parameters of this method in order to achieve the desired solubility within the indicated range. Advantageously, the solubility of the dried pea or fava bean protein at pH 7.0 and at pH 4.0 ranges from 40% to 55%.

[0154] Solubility: test A

[0155] The solubility is determined using the Test A method described below:

[0156] Measurement of solubility in water

[0157] This measurement is based on diluting the sample in distilled water, centrifuging and analyzing the supernatant.

[0158] Procedure :

[0159] Introduce 150 g of distilled water at 20°C ± 2°C into a 400 ml beaker, mix with a magnetic stirrer bar and accurately add 5 g of the sample to be tested.

[0160] Adjust the pH to the desired value with 0.1 N NaOH or HC1 (pH 7) or do not adjust the pH.

[0161] The total water content is 200 g.

[0162] Mix at 1000 rpm for 30 minutes and centrifuge at 3000 g for 15 minutes.

[0163] 25 g of supernatant was collected.

[0164] Introduced into a pre-dried and weighed crystallizer.

[0165] Place in an oven at 103℃±2℃ for 1 hour.

[0166] It was then placed in a desiccator (with desiccant) to cool to ambient temperature and weighed.

[0167] The soluble dry matter content is expressed in % by weight and is given by the following formula:

[0168] [Formula 1]

[0169]

[0170] in:

[0171] P = weight of sample (in g) = 5 g

[0172] m1 = weight of the crystallizing dish after drying (in g)

[0173] m2 = weight of the empty crystallizing dish (in g)

[0174] P1 = weight of sample collected (in g) = 25 g

[0175] According to one embodiment, the dried pea or bean protein may have a gelling capacity. The gelling capacity may be in the range of 1 Pa to 100 Pa, preferably between 10 Pa and 90 Pa, and even more preferably between 30 Pa and 60 Pa.

[0176] Gelling capacity: test B

[0177] The term "gelling ability" refers to a functional property consisting of the ability of a protein composition to form a gel or network, which increases viscosity and creates a state of matter intermediate between a liquid and a solid state. The term "gel strength" may also be used. In order to quantify this gelling ability, it is necessary to generate this network and assess its strength. To perform this quantification, Test B is used in the present invention, which is described as follows:

[0178] 1) Dissolve the protein composition to be tested in water at 15% + / - 2% solids and pH 7 at 60°C ± 2°C;

[0179] 2) Stir at 60℃±2℃ for 5 minutes;

[0180] 3) Cool to 20°C ± 2°C and stir at 350 rpm for 24 hours;

[0181] 4) Suspension is performed using a controlled stress rheometer equipped with concentric cylinders;

[0182] 5) Measure the elastic modulus G' and the viscous modulus G" by applying the following temperature curve:

[0183] a. Stage 1: Parameter G'1 is measured after stabilization at 20°C ± 2°C and heating from 20°C ± 2°C to 80°C ± 2°C within 10 minutes;

[0184] b. Stage 2: Stabilize at 80°C ± 2°C for 110 minutes; c. Stage 3: Cool from 80°C ± 2°C to 20°C ± 2°C within 30 minutes and stabilize at 20°C

[0185] Measure G'2 after stabilization at ±2℃;

[0186] 6) Calculate the gelling capacity which is equal to G'2 - G'1.

[0187] In a preferred embodiment, the controlled stress rheometers are selected from the models DHR 2 (TA, Instruments) and MCR 301 (Anton Paar), which have concentric cylinder spindles. They are equipped with a temperature control system based on the Peltier effect. To avoid evaporation problems at high temperatures, liquid paraffin is added on top of the sample.

[0188] For the purposes of this invention, a "rheometer" is a laboratory machine used to measure the rheological properties of fluids or gels. It applies forces to a sample. Typically, due to its characteristically small size (the mechanical inertia of the rotor is very low), it allows for fundamental studies of the mechanical properties of liquids, gels, suspensions, pastes, etc., in response to applied forces.

[0189] The so-called "controlled stress" model makes it possible to determine the intrinsic viscoelasticity of a material, which depends particularly on time (or angular velocity ω) and temperature, by applying a sinusoidal stress (oscillatory mode). In particular, this type of rheometer makes it possible to obtain the complex modulus G*, which in turn makes it possible to obtain the modulus G' or elastic part and G" or viscous part;

[0190] The first three steps consist of resuspending the protein in water, using precise conditions to maximize subsequent measurements.

[0191] The water chosen is preferably reverse osmosis water, but potable water may also be used.

[0192] Its temperature is 60°C ± 2°C during the initial resuspension (steps 1 and 2), and then 20°C ± 2°C after 24 hours of solubilization and cooling before measurement (step 3). In general, and unless otherwise stated, when a temperature is given in this specification, it always includes a variation of ± 2°C, for example 20°C ± 2°C or 80°C ± 2°C.

[0193] A defined amount of protein is added to the water to obtain a suspension containing 15% ± 2% solids. To achieve this, equipment familiar to those skilled in the art, such as a beaker and a stirring rod, is used. A 50 mL volume is stirred at 350 rpm at room temperature for at least 10 hours. Generally speaking, and unless otherwise indicated, solids contents given in this specification always include a variation of ± 2%, e.g., 15% ± 2%. The pH is adjusted to 7 ± 0.5 using a pH meter and an acid-base reagent, as is known in the art.

[0194] The fourth step consists of introducing the sample into the rheometer and covering it with a thin layer of oil to limit evaporation.

[0195] During the fifth step, the following temperature regime is then applied: a. Stage 1: heating from a temperature of 20°C ± 2°C to a temperature of 80°C ± 2°C within 10 minutes; b. Stage 2: stabilization at a temperature of 80°C ± 2°C for 110 minutes; c. Stage 3: cooling from a temperature of 80°C ± 2°C to a temperature of 20°C ± 2°C within 30 minutes.

[0196] During this protocol, the parameter G' was continuously measured and recorded.

[0197] The sixth and final step of Test B consists of utilizing this record. Two values ​​are extracted: G'1 = G' value at the beginning of stage 1 after stabilization at 20°C ± 2°C, and G'2 = G' value at the end of stage 3 after stabilization at 20°C ± 2°C.

[0198] The gelling power is equal to G'2-G'1.

[0199] According to an optional variation, pea or broad bean protein is an enzymatically modified protein. For enzymatically modified proteins, those skilled in the art refer to proteins whose protein structure is intentionally modified by adding at least one enzyme capable of modifying the protein structure to the protein. The enzyme can be selected from proteases, peptidases and deamidases, such as those of EC 3.5.1 type (such as glutaminases or deiminases), such as those of EC 3.5.3 type (such as peptidyl arginine deiminases). It is well known that these protein modification enzymes are used to change the physicochemical and / or sensory properties of proteins. For example, it is known from document WO2019 / 233920A1 that peptidyl arginine deiminases reduce astringency, especially the astringency of rapeseed protein. If the method includes protein hydrolysis of a protein-rich fraction, this can change the degree of hydrolysis (DH) of the protein. Preferably, the degree of hydrolysis is less than 15%, advantageously less than 10%, preferably less than 6%, for example, between 3% and 5%. Those skilled in the art will know how to adjust the enzymatic protein hydrolysis conditions, or even not perform such a step to obtain the desired DH. According to a preferred embodiment of the present invention, the pea or faba bean protein is not enzymatically modified by deamination. According to another preferred embodiment of the present invention, the protein is not enzymatically modified. One advantage of the present invention is that the organoleptic properties of pea proteins can be modified, and in particular, a milky flavor can be imparted to them, without even requiring enzymatic modification of the protein. According to one embodiment, the present invention can also provide proteins with an unmodified primary structure.

[0200] Use of pea proteins

[0201] Another object of the present invention is the use of the pea or bean protein according to the invention for producing a food or beverage product, in particular a plant-based milk substitute.

[0202] Generally speaking, the pea or faba bean protein of the present invention can be used in food products and beverages that can include an amount of up to 100% by weight relative to the total dry weight of the food or beverage product, for example, an amount of pea protein in the range of about 1% to about 80% by weight relative to the total dry weight of the food or beverage product. All intermediate amounts (i.e., 2%, 3%, 4%, ... 77%, 78%, 79% by weight relative to the total weight of the food or beverage product) and all intermediate ranges based on these amounts can be used. These food and beverage products may be suitable for vegetarians or vegan groups.

[0203] A particularly interesting use of the protein of the present invention relates to its use in beverages having a more pleasant taste than those obtained from other commercially available pea proteins. The pea or faba bean protein of the present invention can advantageously be used to produce beverages, in particular milk substitutes, or in other words milk alternatives. In addition, due to the milky taste characteristic produced by the ingredient, these beverages can also have a richer milky taste characteristic than beverages that do not contain the protein, which is an undeniable advantage for the production of plant-based milk alternatives. In addition to improving the flavor, the present invention also makes it possible to obtain a softer mouthfeel than when using other pea or faba bean proteins, which is advantageous for beverages and in particular for plant-based milk alternatives, since animal milk also generally has a soft mouthfeel.

[0204] In beverages, the protein content of these products may vary greatly, and may also be high-protein beverages. The protein content may range, for example, from 1% to 12% by dry weight relative to the gross weight of the beverage, particularly from 3% to 10% by dry weight relative to the gross weight of the beverage. The beverage may be of any type and may include plant-based alternatives to milk or milk substitutes, including barista-type milk and coffee creamer. These may also include other acidic or non-acidic ready-to-drink beverages, such as 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 water, fruit juice, and sweetened or unsweetened tea or coffee drinks); alcoholic beverages, such as beer or spirits, smoothies, and beverage concentrates (including but not limited to liquid concentrates and syrups, and non-liquid "concentrates," such as freeze-dried and / or powdered preparations or "powder mixes"). It should be noted that in beverages, flavorings or masking agents are typically used to reduce the pea or bean flavor characteristics or bitter aftertaste of protein, or to flavor the beverage. One of the advantages of the pea or faba bean protein of the present invention is that using it in place of conventional pea or faba bean protein allows for a reduction in the amount of flavoring or masking agents used, or even the elimination of these ingredients entirely, while retaining the beverage's highly satisfying taste. These beverages may also contain hydrocolloids; however, because the pea or faba bean protein provides a softer texture, the hydrocolloid content can be reduced or even eliminated while retaining a soft mouthfeel.

[0205] The food products that may be involved include baked products, such as bread products (including but not limited to sourdough and unleavened bread, sandwich bread, yeast bread and unleavened bread, such as baking soda bread), bread containing all types of wheat flour, bread containing all types of flour other than wheat flour (such as potato flour, rice flour, barley flour, spelt flour and rye flour), gluten-free bread; mixtures for preparing said baked products; sweet baked products (including but not limited to rolls, cakes, pies, pastries, waffles, pancakes, muffins, pancakes and cookies); mixtures for preparing said sweet baked products; pie fillings and other sweet fillings (including but not limited to fruit pie fillings and nut pie fillings, such as pecan pie fillings, and fillings for cookies, cakes, pastries, confectionery products, etc., such as cream fillings); and snack bars (including but not limited to energy bars, cereal bars, nut bars and / or fruit bars).

[0206] They can also be jelly desserts, such as dessert creams, custards, and puddings. Another type of dessert can also be 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 frozen non-dairy desserts, such as non-dairy ice cream, sorbet, etc.).

[0207] Other products conventionally prepared using animal milk may also contain the pea or broad bean protein of the present invention to form a substitute. These products may be acidified and / or fermented with a starter culture (such as a lactic acid starter culture, a vegetarian starter culture, or a mesophilic starter culture). These may include yogurt (including but not limited to whole milk yogurt, low-fat yogurt, and skim yogurt, which may be free of milk protein and lactose). The term "yogurt" also includes fresh cheese and small Swiss cheese. It may also include cheese substitutes such as cheese sauces, melted cheeses, pressed ripe cheeses and raw cheeses, soft cheeses, twirled cheeses, blue cheeses; it may include Emmental, string cheese, ricotta, provolone, Parmesan, Munster, mozzarella, Monterey Jack, Manchego, blue cheese, fontina, feta, Edam, double Gloucester, Camembert, cheddar, Brie, Asiago, and Havarti. It can also include other products such as plant-based butter and even crème fraîche.

[0208] Other products that may comprise the pea or bean protein of the invention are also sauces, such as vinaigrettes or mayonnaise or ketchup based sauces or syrups.

[0209] Likewise, the pea or fava bean protein of the present application can be incorporated into confectionery products including, but not limited to, jelly candies, gummies, hard candies, chocolates, caramels, and chewing gums; sweetened and unsweetened breakfast cereals including, but not limited to, extruded cereals, flaked cereals, and puffed cereals; and cereal coating compositions for making breakfast cereals. These can also include spreads including, but not limited to, jellies, jams, nut butters such as peanut butter, sauces, and other spreadable products.

[0210] The pea or fava bean protein of the present application can also be used as a flavor carrier or encapsulating agent.

[0211] Other types of food and beverages not mentioned herein but which typically comprise one or more proteins can also be envisaged in the context of the present application. In particular, animal feed such as pet food is explicitly envisaged.

[0212] The pea or fava bean protein can also optionally be used in a meat substitute such as an emulsified sausage or burger, or in a fish or seafood substitute, after texturization. It can also be used in an egg replacement formulation or for the production of a protein product such as tofu or tempeh. Texturized protein generally refers to protein that is texturized by extrusion (i.e. dry extrusion) or texturized plant protein or high moisture extrusion. The extruder can be single screw, twin screw or multi screw. In the case of twin screw extrusion, the extrusion can be co-rotating or counter-rotating. As an example of multi screw extrusion, planetary and ring extruders can be mentioned. Other more specific technologies can also be cited such as shear cell technology, micro-extrusion or 3D printing.

[0213] The food or beverage is especially useful for special nutrition, for example for specific population groups such as infants, children, adolescents, adults, elderly, athletes, sick population groups, etc. These can be meal replacement formulations or complete nutritional beverages, for example for weight management or clinical nutrition (e.g. tube feeding or enteral nutrition).

[0214] Pea or faba bean protein can be used as a single protein source, but can also be used in combination with other additional plant or animal proteins. These additional proteins can be hydrolyzed or non-hydrolyzed. These additional proteins are usually in the form of concentrates or isolates. Concentrates and isolates are distinguished according to their protein content: concentrates usually have a protein content between 50% and 70%, and isolates have a protein content greater than 70%, preferably between 80% and 90%, respectively. The term "plant protein" refers to all proteins derived from cereals, oil-containing plants, legumes and tuberous plants, as well as all proteins derived from algae and microalgae or fungi, used alone or as a mixture, selected from the same family or different families. In this application, "legumes" generally refers to the dicotyledonous plant family of the order Fabales. Several legumes are important crop plants, such as soybeans, kidney beans (especially mung beans), chickpeas, broad beans, peanuts, cultivated lentils, cultivated alfalfa, various clovers, broad beans, locust beans, liquorice and lupins. Additional legume proteins can be selected from these legumes, or pea or faba bean proteins, such as the pea or faba bean proteins used in the present invention. In this application, the term "cereal" refers to plants cultivated from grasses that produce edible grains, such as wheat, oats, rye, barley, corn, sorghum or rice. Tubers can be carrots, cassava, konjac, potatoes, Jerusalem artichokes, sweet potatoes. Oilseed plants are generally plants that produce seeds from which oil is extracted. Oilseed plants can be selected from sunflower, rapeseed, peanuts, sesame, pumpkin or flax. Animal proteins can be, for example, egg or milk proteins, such as whey protein, casein or caseinate protein. The pea or faba bean protein composition of the present invention can therefore be used in combination with one or more of these proteins or amino acids to improve the nutritional properties of the final product, for example, to improve the PDCAAS of the protein or to provide other functionality.

[0215] Pea or bean proteins can also be used to produce pharmaceutical products or in fermentations, for example to produce fungal metabolites or cell culture metabolites.

[0216] The invention and its advantages will now be illustrated in the embodiments described in detail in the Examples section hereinafter.It should be noted that these examples do not limit the invention.

[0217] Examples

[0218] Example 1 : Pea protein concentrate

[0219] Approximately 1000 kg of peas were used. The outer fibers of the peas were first separated from the seeds by crushing (mechanically separating the hulls from the pea seeds) and dehulling (using compressed air to sort the hulls and dehulled seeds). The seeds thus prepared were ground using a mill to a particle size such that 88% of the particles were smaller than 100 microns and 1.9% were larger than 315 microns. The resulting flour was mixed with water to produce a suspension of ground pea seed flour with a dry matter content of approximately 20%. This suspension of ground pea seeds was stirred for 5 minutes and fed into a decanter centrifuge (Flottweg Z3). A protein-rich fraction (approximately 7% dry matter content) was recovered from the overflow.

[0220] The protein-rich fraction was adjusted to pH 6.6 using sodium hydroxide and hydrochloric acid, then heat-treated at 130° C. for 10 seconds and rapidly cooled to about 60° C. The solution was then atomized on a Nubilosa atomizer (air inlet temperature = 195° C. - air outlet temperature = 95° C.). The pea protein powder, designated "pea concentrate according to Example 1," was then analyzed.

[0221] Table 1

[0222]

[0223]

[0224] ND stands for Not Detected. In the case of measurements, ND means Not Measured.

[0225] The pea concentrate obtained according to the invention is of particular interest since it contains approximately 60% protein based on dry matter and has a solubility of more than 45% at pH 4 or 7.

[0226] It also contains 12.9% GOS, which may be defructosylated according to the teachings of Example 2 to enrich it with this natural fiber.

[0227] The starch content of the pea concentrate obtained according to the invention (wet process) is lower than the starch content of the pea protein concentrate sold by LA-VITA under the name "Pea Protein Concentrate" and the tested starch content of the VESTKORN pea protein concentrate obtained by the dry process.

[0228] The combination of the content and quality of proteins (globulins + albumins) and the presence of natural soluble fibers makes it a preferred food ingredient.

[0229] Example 2: Defructosylation reaction of pea proteins with invertase :

[0230] Proceed as in Example 1, but before the pH neutralization step, a defructosylation step was performed on the protein fraction recovered from the overflow of the decanter centrifuge (Flottweg Z3).

[0231] The pH of the resulting protein-rich fraction was adjusted to 5.0 using hydrochloric acid and sodium hydroxide. The rectified protein-rich fraction was heated and then temperature-controlled at 60° C. 0.2% by dry weight of the enzyme Sumizyme INV (invertase) was added and allowed to react for 40 minutes under stirring.

[0232] The remainder of the process was identical to Example 1. The pH was then neutralized to 6.6. The solution was evaporated to 20% dry matter, subjected to HTST treatment at 130°C for 10 seconds, and then rapidly cooled to 60°C upon exiting. The solution was then atomized on a Nubilosa atomizer (air inlet temperature = 195°C - air outlet temperature = 95°C). The pea protein powder, designated "Defructosylated pea concentrate according to Example 2," was then analyzed.

[0233] Table 3

[0234]

[0235] GOS (italicized, about 12%) are hydrolyzed into defructosylated GOS (about 4.5%) and fructose (6%). Fructose provides a sweet flavor that softens the bitter taste of pea protein.

[0236] It should be noted that the enzymatic treatment with invertase may be performed further downstream, namely on the concentrate obtained in step 1 .

[0237] Example 3: Defructosylation reaction of pea proteins by fermentation:

[0238] Proceed as in Example 1, but before the pH neutralization step, a defructosylation step was performed on the protein fraction recovered from the overflow of the decanter centrifuge (Flottweg Z3).

[0239] The recovered protein fraction was fermented using the Bacillus subtilis strain deposited at the CNCM under the reference CNCM 1-5515.

[0240] Use containing 10 8 A 5 ml cryovial of UFC / ml was inoculated into a 2 l erlen with baffles containing 500 ml of LB medium (10 g / l of BactoTrypton, 5 g / l of BactoYest extract, and 10 g / l of sodium chloride (NaCl), sterilized at 120°C for 20 minutes). The erlen was incubated at 37°C while stirring at 150 RPM for 4.5 hours.

[0241] The protein fraction was evaporated to reach a dry matter content of 20%. A defructosylation fermentation was then performed in a 15 L fermenter after inoculation with the 10% preculture obtained in paragraph 169 of the preceding section. The fermentation parameters were as follows: air flow rate of 0.25 VVM, no p02controlled directly in the liquid medium. Stirring was set to 300 RPM. The pH was adjusted to 6.5 with sodium hydroxide and hydrochloric acid, but no adjustment was made thereafter.

[0242] The rest of the process was almost identical to Example 1, except for the concentration step that had already been performed. The pH was then neutralized to 6.6. A HTST (high temperature short time) treatment was then performed at 130°C for 10 seconds, with a rapid cooling at 60°C when leaving. The solution was then atomized on a Nubilosa atomizer (air inlet temperature = 195°C - air outlet temperature = 95°C).

[0243] The analysis showed that GOS (raffinose, stachyose, verbascose) were hydrolyzed into defructosylated GOS (manninotriose, melibiose). Fructose was consumed by the strain, which distinguishes this alternative from the alternative with invertase (Example 2).

Claims

1. A method for producing pea or broad bean protein, the method comprising the following steps:

1. preparing an aqueous suspension of ground pea or bean seeds in an aqueous solution, optionally in the presence of a heat treatment; 2. removing the insoluble fraction by solid / liquid separation of the aqueous suspension of ground pea or bean seeds obtained in step 1) to obtain a protein-rich fraction; 3. Defructosylation of galacto-oligosaccharides from the protein-rich fraction obtained in step 3) by enzymatic and / or fermentative means.

2. The method according to claim 1, characterized in that Said aqueous suspension of pea or bean seeds of step 1 is obtained by adding pea or bean flour obtained by dry grinding before dispersing said pea or bean flour in the aqueous solution.

3. The method according to claim 1, characterized in that When the pea or bean seeds are introduced into the aqueous solution as whole pea or bean seeds, step 1) of the method comprises a step of wet grinding the aqueous composition formed between the pea or bean seeds and the aqueous solution so as to obtain the aqueous suspension of ground pea or bean seeds.

4. The method according to claim 1 , wherein The aqueous suspension of ground pea or bean seeds of step 1), the optional heat treatment of step 1 comprising: a) introducing pea or bean seeds or ground pea or bean seeds into an aqueous solution at a temperature between 65° C. and 90° C. so as to obtain an aqueous composition comprising pea or bean seeds or ground pea or bean seeds; b) subjecting said aqueous composition obtained in step a) formed between said pea or bean seeds or said ground pea or bean seeds and said aqueous solution to a heat treatment at a temperature comprised between 40° C. and 65° C. for a period of 1 to 10 minutes.

5. The method according to claim 1 , wherein The defructosylation of galacto-oligosaccharides (GOS) in step 3 is performed using an enzyme selected from the group consisting of invertase, α-galactosidase, β-fructosidase and any combination thereof.

6. The method according to claim 1, wherein Said defructosylation of galacto-oligosaccharides (GOS) is carried out with a microorganism of the genus Bacillus, preferentially Bacillus subtilis, preferentially a strain of Bacillus subtilis, as deposited with the CNCM on May 28, 2020 under number I-5515.

7. A composition of pea or broad bean protein, characterized in that It comprises a protein content expressed in grams of protein per 100 g of dry matter of between 50% and 70%, said protein consisting of a mixture of globulins and albumins; and between 3% and 15% of soluble fibres derived from pea or bean galacto-oligosaccharides.

8. The composition according to claim 7, characterized in that The globulin / albumin dry weight ratio is between 70:30 and 90:10, preferably between 75:25 and 85:

15.

9. The composition according to claim 7, characterized in that The soluble fiber derived from pea or bean galacto-oligosaccharides is selected from the list comprising melibiose, mannotriose, verbascotetraose and mixtures thereof.

10. Use of the composition of pea or faba bean proteins according to one of claims 7 to 9 or obtained according to one of claims 1 to 6 for the production of a food or beverage product, in particular a plant-based milk substitute.

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