Method for preparing a plant protein isolate by fermentation

Abstract

The invention relates to a method for preparing a protein isolate, comprising the following steps: providing a seed; micronizing the seed, to obtain a micronized fraction; treating the micronized fraction by aeraulic separation, to collect a starch-enriched fraction; mixing the starch-enriched fraction with an aqueous liquid, to form a liquid substrate; hydrolyzing the starch in the liquid substrate, to obtain a hydrolyzed substrate; fermenting the hydrolyzed substrate, to obtain a fermented medium; distilling the fermented medium, to collect a distillate and a distillation residue; treating the distillation residue via a solid-liquid separation; collecting the solid fraction; the method further comprising bringing the liquid or hydrolyzed substrate and / or the distillation residue into contact with at least one enzyme selected from xylanases, glucanases, galactosidases and combinations thereof.

Description

[0001] Method for preparing a plant protein isolate by fermentation

[0002] Scope of the invention

[0003] The present invention relates to a method for producing a plant protein isolate from starch-containing plants, such as legumes or cereals.

[0004] Technical background

[0005] Global demand for protein is growing rapidly, driven by population increases across all continents and greater prosperity, particularly in Asia and Africa. However, the recent and increasingly severe climate crises are forcing governments to implement proactive environmental policies to reduce greenhouse gas emissions.

[0006] Proteins currently come almost entirely from animal sources. Raising cattle, sheep, and poultry requires significant water consumption and generates substantial greenhouse gas emissions. In contrast, plant-based protein production has a considerably less negative environmental impact.

[0007] The transition from animal to plant-based proteins is not straightforward. Animal products are preferred due to their taste, which is partly linked to the microstructure of meat and its nutritional composition. Plants that produce proteins also contain carbohydrates and other molecules that influence taste and nutritional profile. It is these characteristics specific to the plant world that have fostered the development of processes for fractionating plant products.

[0008] Among plant-based protein sources, three classes are most commonly used: cereals (especially wheat and corn), followed by oilseeds (notably soybeans and sunflowers), and then legumes. Historical methods of extracting protein from plants are known as wet processes, which vary depending on the plant class.

[0009] In the case of wheat, which contains gluten as a vital protein source, extraction begins with a series of millings to separate the fibers (bran) from the flour. The flour is then mixed with water in a mixer to form a dough and thus develop the gluten. This mixture is then sent to a three-phase decanter, from which three separate fluids are extracted: gluten, pentosans, and starch. The gluten is then washed in rotating drums, drained, and dried. The amount of water used in this process is generally between 2.5 and 3.5 m³. 3per tonne of wheat. Therefore, there is a double negative environmental impact:

[0010] - The first is related to the use of significant quantities of water. For example, a medium-sized starch factory uses approximately 2,000 tonnes of wheat per day, which corresponds to a withdrawal of about 2 million m³ 3 of water per year.

[0011] The second factor is linked to the intensive use of fertilizers for wheat cultivation, its transport, and all the energy required for the gluten extraction process, which notably includes the evaporation of water consumed upstream. The environmental impact in greenhouse gases estimated by the CarbonCloud platform is 2,300 kg CCheq / t of wheat gluten produced.

[0012] The process of extracting gluten from corn begins with soaking the grain to facilitate milling. Subsequent steps utilize various separation methods (filter screens, centrifuges, hydrocyclones) and result in three products: gluten, the oil-rich germ, and starch. The gluten is concentrated using a decanter or a rotary vacuum filter and then dried. Similar to wheat, this process also has a double negative environmental impact, linked to:

[0013] - water consumption, which is estimated at 1.4 m 3 per ton of corn. For a medium-sized starch factory that grinds 1,500 tons of corn per day, this represents a withdrawal of approximately 700,000 m³ 3 of water per year.

[0014] - The use of fertilizers and the energy required for water evaporation result in a greenhouse gas impact of 1,700 kg CCHEq per tonne of corn gluten produced (source: CarbonCloud). In the case of oilseeds such as soybeans and sunflowers, industrial-scale fractionation is preceded by the extraction of fats using a solvent, primarily hexane. The residue insoluble in hexane is rich in protein and fiber and is called oilcake. The oilcake obtained after oil extraction is then dried. The environmental impact is therefore mainly linked to crop cultivation, the use of hexane, and the energy expended for oil extraction and oilcake drying. The greenhouse gas impact is estimated, for soybeans, at between 1,000 and 2,000 kg CCHEq per tonne of soybeans used, and for sunflowers, at between 2,000 and 3,000 kg CCHEq per tonne of sunflowers used.

[0015] With regard to legumes, the protein extraction process first involves a step of grinding the seed to obtain flour.

[0016] In one protein extraction method, ground seeds are suspended in an alkaline solution with a pH close to 9. Proteins from the globulin and albumin families (which typically represent 75–85% by weight of the total protein) are solubilized, while other components such as starch and fiber remain insoluble. The solids are then separated from the liquids by centrifugation or decantation. The pH of the supernatant (liquid fraction) obtained in the previous step is adjusted to the isoelectric point of the proteins, for example, using hydrochloric acid, this pH often being between 4 and 5. At this pH, the interactions between the proteins and the solvent are reduced, allowing the proteins to be suspended. A further solid-liquid separation is then performed to recover the solid phase. After resuspension, the pH can be adjusted to neutral before the proteins are dried.With this process, proteins of the glutelin family are not extracted because the pH that would have to be applied for their extraction would cause loss of functionality of the albumins and globulins.

[0017] Another approach for processing legumes involves the use of filtration membranes. In this process, the legume seed is ground into a flour. This flour is then suspended at a pH of 7.5 to achieve a dry matter content of 10 to 20% by weight. By centrifugation or decantation, the solid mass containing starch and fiber is separated from the proteins solubilized in the solvent. The supernatant containing the proteins is filtered through organic membranes with a pore size of 10 kDa. Proteins with a high molecular weight are retained in the recirculation loop and collected in the retentate. The smaller molecules that pass through the membrane are collected in the permeate. The retentate then undergoes a diafiltration step, which involves filtering the diluted proteins again with demineralized water.This step aims to "wash" the proteins to minimize the presence of salts and amino acids. The proteins are then concentrated to approximately 30% by dry weight and dried using a spray dryer.

[0018] The techniques used for legume protein extraction described above are very energy-intensive, resulting in a significant environmental impact. The greenhouse gas emission rate is typically between 4,000 and 5,000 kg CCheq per tonne of protein isolate produced (source: CarbonCloud). However, the most significant impact stems from the high consumption of water and chemicals, and consequently, the volume of wastewater requiring treatment.

[0019] Other methods for producing protein fractions have been described.

[0020] For example, the article by Pelgrom et al., "Dry fractionation for production of functional pea protein concentrates," Food Research International, 2013, vol. 53, pp. 232–239, describes a process for preparing a pea protein concentrate comprising a step of grinding unhulled peas, a sieving step, and an air separation step of the ground peas, allowing the production of a fraction of larger particles and a fraction of finer particles, enriched in protein. The resulting protein concentrates contain 51–55% protein (on a dry matter basis).

[0021] US patent 9,476,068 discloses a process for preparing a co-product from plant material comprising starch and another polysaccharide selected from cellulose and hemicellulose, comprising hydrolysis of the starch and / or the other polysaccharide at a temperature of at least 85°C, contacting the starch with an alpha-amylase, contacting the other polysaccharide with a pre-conversion enzyme selected from xylanases, cellulases and hemicellulases, combining the medium with a yeast to carry out a first fermentation, distilling the fermentation mixture and recovering the distillation residue to obtain a first co-product.This first co-product is subjected to a further process including acidification, hydrolysis at a temperature of at least 85°C, the addition of a pre-conversion enzyme, the addition of yeast to carry out a second fermentation, and a second distillation, to obtain a second co-product corresponding to the second distillation residue. The co-products obtained contain approximately 30% protein by weight on a dry matter basis.

[0022] US patent 9,644,228 relates to a process for producing a protein concentrate from a starch-containing seed, comprising grinding the seed, mixing the ground seed with water, mixing the resulting suspension with enzymes to solubilize the starch and glucans, adding a fermentation organism, fermenting the medium, separating the fermentation medium into a solid fraction and a liquid fraction by centrifugation or filtration, collecting and drying the solid fraction, and distilling the liquid fraction. In the examples, the protein concentrates produced contain up to 58% protein by weight.

[0023] These processes allow the production of fractions with an insufficient protein content on a dry matter basis.

[0024] There is therefore a real need to develop a process for obtaining a protein isolate from plant material with a higher protein content and lower greenhouse gas emissions.

[0025] Summary of the invention

[0026] The invention relates primarily to a method for preparing a protein isolate, comprising the following steps:

[0027] - the supply of at least one seed comprising starch and proteins;

[0028] - the micronization of said at least one seed, so as to obtain a micronized fraction;

[0029] - the treatment of the micronized fraction by air separation, in order to collect a fraction enriched in starch;

[0030] - mixing the starch-enriched fraction with an aqueous liquid, so as to form a liquid substrate;

[0031] - the hydrolysis of starch in the liquid substrate, so as to obtain a hydrolyzed substrate;

[0032] - the fermentation of the hydrolyzed substrate, in order to obtain a fermented medium;

[0033] - the distillation of the fermented medium, so as to collect a distillate and a distillation residue; - the treatment of the distillation residue by solid-liquid separation, so as to obtain a liquid fraction and a solid fraction; and

[0034] - the collection of the solid fraction; the process further comprising a step of bringing the liquid substrate or the hydrolyzed substrate and / or the distillation residue into contact with at least one enzyme selected from the group consisting of xylanases, glucanases, galactosidases and combinations thereof.

[0035] In embodiments, at least one seed is chosen from the group consisting of legume seeds, preferably chosen from the group consisting of beans, peas, broad beans, lentils, chickpeas, lupins, field beans and mixtures thereof; cereal seeds, preferably chosen from the group consisting of oats, barley, wheat, maize and mixtures thereof; and combinations thereof.

[0036] In some embodiments, starch hydrolysis includes contacting the liquid substrate with at least one enzyme selected from the group consisting of saccharidases, preferably at least one α-amylase.

[0037] In some embodiments, the hydrolysis of starch includes the gelatinization of the liquid substrate, so as to obtain a gelatinized substrate, preferably by heating said liquid substrate, more preferably the heating being carried out by mixing steam with the liquid substrate.

[0038] In some embodiments, at least one seed includes a film, and the process includes a step of de-filming the seed, prior to micronization.

[0039] In embodiments, the hydrolysis of starch includes a step of contacting the liquid substrate with at least one enzyme selected from the group consisting of glucosidases, preferably with at least two enzymes selected from the group consisting of glucosidases, more preferably with at least one α-1,4-glucosidase and one amylo-α-1,6-glucosidase.

[0040] In some embodiments, the liquid substrate or hydrolyzed substrate and / or distillation residue are contacted with at least one xylanase and at least one glucanase, preferably at least one beta-glucanase; or with at least one glucanase, preferably at least one beta-glucanase, and with at least one galactosidase, preferably at least one alpha-galactosidase. In some embodiments, the aerodynamic separation of the micronized fraction is carried out using a cyclone separator.

[0041] In embodiments, solid-liquid separation is chosen from separations by centrifugation, filtration, decantation and combinations thereof.

[0042] In some embodiments, the process includes, for carrying out the fermentation, bringing the liquid substrate or the hydrolyzed substrate into contact with at least one microorganism, preferably chosen from the group consisting of yeasts, bacteria and combinations thereof, more preferably at least one microorganism is at least one yeast.

[0043] In some embodiments, the process includes a step of drying the solid fraction.

[0044] In embodiments, the protein isolate comprises at least 70% by weight, preferably at least 75% by weight, of protein, relative to the total dry weight of the protein isolate.

[0045] In some embodiments, a fermentation product is produced during fermentation, said fermentation product being chosen from among alcohols, and preferably being ethanol.

[0046] The present invention addresses the need expressed above. More specifically, it provides a more environmentally friendly process for preparing a protein isolate from plant material, particularly one with low greenhouse gas emissions, that is easily implemented on an industrial scale while still yielding a high protein content. Furthermore, the process according to the invention allows for the production of other products of interest, such as ethanol or other organic compounds.

[0047] This is achieved through the implementation of a particular sequence of steps including micronization of the plant material, aeraulic separation, fermentation, distillation and solid-liquid separation of the distillation residue combined with hydrolysis by means of deviscosifying enzymes (in particular xylanases, glucanases and / or galactosidases).

[0048] Furthermore, in embodiments where the plant material is a legume, the process according to the invention allows for an even further reduction in greenhouse gas emissions. Indeed, legumes have the ability to utilize atmospheric nitrogen for their growth, through symbiotic organisms that release this atmospheric nitrogen into the soil. This ability allows for a reduced use of nitrogen-based chemical fertilizers, which are sources of greenhouse gases.

[0049] Detailed description

[0050] The invention is now described in more detail and in a non-limiting manner in the following description.

[0051] Unless otherwise stated, all percentages are mass percentages.

[0052] In this text, the quantities indicated for a given species may apply to that species according to all its definitions (as mentioned in this text), including more restricted definitions.

[0053] The invention relates to a process for producing a protein isolate from at least one plant material comprising starch and proteins. By "protein isolate" is preferably understood to mean a fraction comprising at least 70% by weight of protein, more preferably at least 75% by weight of protein, and more preferably at least 78% by weight, relative to the total dry weight of the fraction.

[0054] Preferably, the plant material also includes fiber. "Fiber" is defined as all plant polymeric molecules, soluble or insoluble, other than starch and starch fragments. Fibers include, in particular, cellulose, hemicellulose, lignin, 3-glucans, and pectin. The plant material may also include lipids (or fats).

[0055] Preferably, the plant material comes from at least one legume, at least one cereal, or combinations thereof.

[0056] The term "legume" refers to plants of the Fabaceae family. Legume seeds contain, among other things, starch and protein. They have the advantage of being rich in carbohydrates (they can contain approximately 60% carbohydrates), primarily in the form of starch, and rich in protein (they generally contain between 20 and 30% protein by weight on a dry matter basis), and of having a low fat content.

[0057] All legumes are suitable for the invention. Examples of legumes usable in the invention include, in particular, beans, peas, broad beans, lentils, chickpeas, lupins, field beans, and mixtures thereof. Cereals belong to the Poaceae family. They produce seeds, also called grains, which contain starch and proteins. The plant material can come from any suitable cereal. Examples of cereals usable in the invention include, in particular, wheat, maize, barley, oats, and mixtures thereof.

[0058] Advantageously, the plant material according to the invention comprises at least one seed. More advantageously, the plant material is at least one seed.

[0059] Generally, seeds comprise at least one kernel surrounded by a hull (or husk). However, in this text, the term "seed" may generally refer to the whole seed as well as any part of the seed (for example, the kernel), unless otherwise specified.

[0060] The seeds used in the invention as starting plant material preferably comprise a skin and a kernel.

[0061] Even more advantageously, the plant material includes, or is, at least one legume seed. Using legumes as a raw material is beneficial because their cultivation results in low greenhouse gas emissions. Indeed, legumes are the only plants capable of fixing atmospheric nitrogen in the soil through their symbiotic association with Rhizobium bacteria via the formation of nodules, thus providing the plant with the nitrogen necessary for its growth. The ability of legumes to fix atmospheric nitrogen eliminates the need for nitrogen fertilizers, which, when applied in excess, harm soil biodiversity and therefore its fertility. Furthermore, the application of nitrogen fertilizers releases a large quantity of nitrous oxide, a greenhouse gas.Furthermore, the atmospheric nitrogen fixed by legumes is returned to the following crop through the decomposition of crop residues (above and below ground) by Rhizobium bacteria. The most readily degradable residues (leaves, stems with low woody fibers and a low carbon-to-nitrogen ratio) decompose and release nitrogen within a few weeks, while the woody parts (stems, roots) mineralize more slowly. Specifically, carbon emissions associated with legume cultivation are estimated at 200 kg CCheq per tonne of legume.When legume cultivation is associated with cereal crops in a crop rotation process (e.g., alternating pea, wheat and oat crops), the input of nitrogen fertilizers is reduced, which can reduce carbon emissions by 189 kg CCheq per tonne of legume: the legume crop therefore has in this case a net almost neutral balance of 11 kg CCheq per tonne of legume.

[0062] Preferably, the plant material comprises, or is, a pea seed.

[0063] Plant material, particularly seeds, may include a hull. Advantageously, the process according to the invention includes a step of removing the hull (or dehulling) from the seed. Indeed, the seed hull is composed mainly of insoluble fibers that are not consumed by fermentation microorganisms. Furthermore, since the majority of seed contaminants are found in the hull, its removal reduces the risk of contamination of the prepared isolate.

[0064] Preferably, de-skinning is a mechanical de-skinning, carried out for example by abrasion, compression, impact, shearing or any other appropriate mechanical action.

[0065] In some embodiments, dehulling is carried out by grinding the seed and then separating the particles obtained according to their size and / or density.

[0066] For grinding, any suitable type of grinder can be used, particularly any grinder that utilizes one of the mechanical forces mentioned above. In particular, a roller mill (also called a cylinder mill) using compressive force can be used. The roller gap is preferably 3 to 6 mm, and even more preferably 4 to 5 mm. After grinding, a mixture of skin fragments and almond pieces is obtained.

[0067] The separation stage of the particles obtained can be carried out in particular by sieving, by air separation, or by a combination of these two techniques.

[0068] The separation step is preferably carried out by sieving followed by air separation.

[0069] Thus, the particles obtained after grinding can undergo one or more sieving stages. In particular, the particles can be separated by passing them through a sieve with a suitable mesh size, allowing for the separation of larger skin fragments and kernel pieces from smaller ones (resulting in two particle fractions of different sizes). Specifically, at least one sieving can be performed using a sieve with a mesh size between 500 µm and 8 mm, preferably between 1 and 6 mm, for example, between 2 and 5 mm. The particles can be subjected to several sievings with different mesh sizes to obtain several particle fractions (comprising skin fragments and kernel pieces) of different sizes.For example, particles larger than 5 mm can be separated from particles between 2 and 5 mm in size, which themselves can be separated from particles smaller than 2 mm in size.

[0070] Particles containing film fragments and kernel pieces, preferably at least one of the fractions obtained after sieving, and more preferably each fraction obtained by sieving, can be separated by air separation. The film fragments (lighter) are carried by the airflow, while the kernel pieces (heavier) are collected at the other end of the device. The applied gas flow rate (preferably air) is preferably between 1500 and 3500 m 3 / h, preferably still at 2000 to 3000 m 3 / h.

[0071] Optionally, when the plant material is a cereal seed, the process may include a step of deburring (or awn removal) of the seed, carried out for example by abrasion, more particularly by means of a deburrer, this step being preferably carried out before hulling.

[0072] The plant material, optionally dehulled and optionally in pieces, undergoes a micronization step, and a micronized fraction is collected. Advantageously, the micronization is a dry process.

[0073] "Micronization" refers to a grinding process that produces particles with a volume median diameter of less than 100 µm, preferably less than 50 µm, and even more preferably less than 30 µm. The volume median diameter (D50) of the particles can be measured according to standard NF ISO 13320-1.

[0074] Micronization can be carried out by any suitable mill (in particular, any mill using mechanical forces of abrasion, compression, impact, or shear). For example, a mill using impact force can be used.

[0075] Micronization is most preferably carried out at room temperature (i.e., between 15 and 30 °C).

[0076] Micronization is preferably carried out at a mill speed of 2000 to 3000 rpm, preferably even more so at 2500 to 3000 rpm.

[0077] Micronization has the added advantage of producing low greenhouse gas emissions, preferably carried out at ambient temperature and using a dry process. The process according to the invention includes a purification step for the micronized fraction. Advantageously, this purification includes a step for separating the starch granules from the proteins.

[0078] Following purification, a starch-enriched (and protein-depleted) fraction is collected. A protein-enriched (and starch-depleted) fraction is also preferably recovered. The term "starch-enriched fraction" refers to a fraction in which the ratio of starch to protein molar proportions (on a dry matter basis) is greater than that of the purified fraction. The term "protein-enriched fraction" refers to a fraction in which the ratio of protein to starch molar proportions (on a dry matter basis) is greater than that of the purified fraction.

[0079] Given the size difference between proteins and starch granules (D50 of approximately 1 to 5 µm for proteins and approximately 10 to 30 µm for starch granules) and density, separation based on a difference in particle size, density, or weight is advantageously used. Most preferably, the separation is aerodynamic. "Aerodynamic separation" is defined as any separation technology using a jet of gas (preferably air) that carries at least some of the particles to be separated. More preferably, the separation is cyclonic. It can be carried out using a cyclone, advantageously combined with at least one selector, and more advantageously with at least two selectors. "Selector" is defined as any rotating element with variable speed and equipped with a slotted cylinder installed in a portion (preferably the upper portion) of a cyclonic separator.This equipment increases the efficiency of particle separation based on density. Using an air separation device allows for the recovery of lighter particles, carried by the gas flow (preferably air), at one end of the device (protein-enriched fraction), while heavier particles are collected at the other end (starch-enriched fraction). Preferably, these lighter particles are carried by a vertical gas flow towards the top of the device, and the heavier particles, not carried by the gas flow, fall towards the bottom of the vertical gas flow.

[0080] Air separation is preferably carried out using a selector operating at a rotational speed between 1000 and 3000 rpm, preferably between 1500 and 2000 rpm. The gas flow (preferably air) passing through the selector preferably has a flow rate between 4000 and 6000 m³ / s 3 / h, preferably still between 5000 and 5500 m 3 / h. Preferably, the selector is followed by a second selector operating preferably at a rotational speed between 2500 and 4500 rpm, and even more preferably between 3000 and 3500 rpm. The gas flow rate (preferably air) of the second selector is preferably between 2500 and 5000 m 3 / h and preferably between 3500 and 4000 m 3 / h. When the aerodynamic separation is carried out using several successive selectors, the protein-enriched and starch-enriched fractions according to the invention are the fractions as separated by the last selector.

[0081] During air separation, components other than starch and protein, such as fiber, fat, and ash, tend to be carried into the protein-enriched fraction rather than the starch-enriched fraction, with this tendency being even more pronounced for fiber. The starch-enriched fraction is therefore purified of some of these components. This is achieved through a micronization step prior to air separation, which facilitates the transfer of fiber and ash to the protein-enriched fraction. Indeed, using coarser grinding instead of micronization would lead to a very incomplete separation of proteins, starch, fiber, and ash during air separation, resulting in a starch-enriched fraction that is significantly less purified of fiber and ash.

[0082] The advantage of this method of obtaining the fractions, through micronization followed by air separation, is its low greenhouse gas emissions and the fact that it does not use water. Furthermore, it yields a fraction (enriched in starch) that is depleted in fiber, and to a lesser extent in salts and fats. Consequently, the protein purity of the protein-enriched fraction (which thus contains fiber, fats, and salts) generally cannot exceed 60 to 65% on a dry matter basis.

[0083] Thus, the process according to the invention has the advantage, compared to a process for producing a protein fraction based on recovering the protein-enriched fraction, as described in the article by Pelgrom et al. mentioned above, of allowing a higher protein content. Furthermore, the protein-enriched fraction has the additional disadvantage of representing only a small portion of the total micronized fraction (typically 25 to 30% by volume), with some of the protein being recovered in the starch-enriched fraction. The starch-enriched fraction advantageously represents 60 to 80% by volume, preferably 70 to 75% by volume, of the volume of the micronized fraction obtained after the micronization step.

[0084] Preferably, the starch-enriched fraction comprises an amount of digestible carbohydrates greater than or equal to 40% by weight, preferably an amount of 40 to 90% by weight, preferably a further amount of 50 to 80% by weight, and more preferably 60 to 80% by weight (relative to the total dry weight of the fraction). In particular, the amount of digestible carbohydrates in the recovered starch-enriched fraction may comprise 40 to 50% by weight, or 50 to 60% by weight, or 60 to 65% by weight, or 65 to 70% by weight, or 70 to 75% by weight, or 75 to 80% by weight, or 80 to 90% by weight, relative to the total dry weight of the fraction. By "assimilable carbohydrates" we mean all glucose and oligosaccharides and polysaccharides which are polymers of glucose linked by α(1-4) or α(1-6) bonds, including starch and dextrins.

[0085] Preferably, the starch-enriched fraction comprises a protein content of 30% or less by weight, preferably 0.5% to 30% by weight, more preferably 3% to 20% by weight, and more preferably 5% to 15% by weight (relative to the total dry weight of the fraction). In some embodiments, the protein content in the recovered starch-enriched fraction may be, relative to the total dry weight of the fraction, 0.5% to 3% by weight, or 3% to 5% by weight, or 5% to 7% by weight, or 7% to 10% by weight, or 10% to 12% by weight, or 12% to 15% by weight, or 15% to 20% by weight, or 20% to 30% by weight.

[0086] Preferably, the starch-enriched fraction comprises an amount of fiber less than or equal to 10% by weight, preferably an amount of 0.5 to 10% by weight, preferably again 1 to 6% by weight, relative to the total dry weight of the fraction; in particular the fraction may comprise an amount of fiber of 0.5 to 2% by weight, or 2 to 4% by weight, or 4 to 6% by weight, or 6 to 8% by weight, or 8 to 10% by weight, relative to the total dry weight of the fraction.

[0087] Preferably, the starch-enriched fraction collected comprises an amount of fat (lipids) less than or equal to 5% by weight, preferably an amount of 0.5 to 5% by weight, preferably still 0.5 to 3% by weight, relative to the total dry weight of the fraction; in particular the fraction may comprise an amount of fat of 0.5 to 1% by weight, or 1 to 2% by weight, or 2 to 3% by weight, or 3 to 4% by weight, or 4 to 5% by weight, relative to the total dry weight of the fraction.

[0088] The amounts of protein, digestible carbohydrate, fat and fiber can be determined as indicated below in the Examples section.

[0089] Advantageously, the starch-enriched fraction is then mixed with an aqueous liquid to form a liquid substrate, more particularly a starch-containing fluid or starch fluid. The aqueous liquid is preferably water. The starch fluid is preferably in the form of a dispersion, and even more preferably, it is a starch milk. "Starch milk" refers to a suspension of starch in water (this suspension may include other components, whether or not they are dissolved in water).

[0090] In this text, the term "liquid substrate" refers to the medium obtained by mixing the starch-enriched fraction with the aqueous liquid, as well as the substrate obtained following any further treatment of the medium up to the fermentation stage (in particular, dextrinization, gelatinization, saccharification or, more generally, hydrolysis treatments, as described below).

[0091] Preferably, the liquid substrate (the starch fluid, preferably starch milk) comprises a dry matter content of 10 to 50% by weight, preferably 20 to 40% by weight, more preferably 25 to 35% by weight, for example 10 to 15%, or 15 to 20% by weight, or 20 to 25% by weight, or 25 to 30% by weight, or 30 to 35% by weight, or 35 to 40% by weight, or 40 to 45% by weight, or 45 to 50% by weight.

[0092] The process according to the invention includes a step of starch hydrolysis (also called a liquefaction step) of the liquid substrate. A hydrolyzed substrate is obtained as a result of the hydrolysis. Advantageously, the starch hydrolysis comprises one or more of the following steps: dextrinization, gelatinization, and saccharification, described in more detail below. Preferably, the hydrolysis comprises at least one dextrinization step, at least one gelatinization step, and at least one saccharification step.

[0093] Advantageously, at least one enzyme is introduced into the liquid substrate (preferably into the starch fluid or the gelatinized substrate as described below). The enzyme is preferably a saccharidase, and more particularly an α-amylase. Most preferably, the enzyme is thermostable, especially at temperatures of 55 to 130°C. The amount of enzyme added is preferably 0.5 to 3 kg of enzyme per tonne of dry matter of digestible carbohydrates, and even more preferably 1.0 to 2.5 kg of enzyme per tonne of digestible carbohydrates. The amount of digestible carbohydrates can be measured as indicated in the Examples section below. The pH of the liquid substrate is preferably adjusted to a pH between 3.5 and 6.5, more particularly between 4.0 and 6.0. This pH range allows for optimal enzyme efficiency. α-Amylases are enzymes capable of hydrolyzing starch into dextrins.

[0094] The liquid substrate (preferably starch fluid) is preferably subjected to a starch gelatinization step. The purpose of this step is to cause the starch granules to burst, releasing the starch molecules into the liquid and thus enabling the action of enzymes (which otherwise cannot access the starch molecules enclosed within the granules). The bursting of the starch granules is preferably achieved by heating the liquid substrate (preferably starch fluid) to a temperature that allows liquid to be introduced into the granule, causing it to swell and then burst. The resulting gelatinized substrate is then obtained.

[0095] Gelatinization can be achieved by any type of heating.

[0096] Gelatinization is advantageously carried out by mixing steam with a liquid substrate (preferably starch fluid). Even more preferably, the mixing of steam with the liquid substrate is carried out using a direct steam injection device.

[0097] Preferably, the liquid substrate (preferably, the starch fluid) is heated (preferably by mixing with steam) to a temperature (called in this text the gelatinization temperature) of 50 to 130 °C, preferably 50 to 100 °C.

[0098] A particularly advantageous method is gelatinization, achieved by mixing a stream of water vapor with a stream of the liquid substrate (preferably starch fluid). "Stream" refers to a moving fluid (gas or liquid).

[0099] The mixing of the flows is more preferably carried out continuously, that is to say that the introduction of at least one fluid to be mixed, and preferably of both fluids, into the mixer is carried out at least in part simultaneously with the discharge of the mixer of said mixture.

[0100] Mixing water vapor with the liquid substrate (preferably starch fluid) in a stream allows for a very rapid, even near-instantaneous, temperature rise of the liquid substrate. Compared to using tanks (generally 300 to 500 m³), ​​this method offers significantly faster heating. 3For a residence time of 1 to 2 hours, equipped with steam injection lances, the continuous flow mixing of fluids allows for faster heating, reduced steam consumption, and lower energy consumption. It is estimated that greenhouse gas emissions can be reduced by approximately 40%. Therefore, implementing the gelatinization step by continuously mixing steam with the liquid substrate in a flow allows for an even greater reduction in greenhouse gas emissions from the process.

[0101] In a particularly preferred manner, gelatinization is carried out using a continuous direct steam injection device, preferably a jet-cooker (or cooker) device.

[0102] However, in other embodiments, gelatinization can be carried out in a tank or other reactor, particularly using steam injection lances or any other suitable steam injection device. In these embodiments, gelatinization can last from 15 minutes to 3 hours, preferably from 20 minutes to 2 hours.

[0103] The gelatinized substrate can be used as a fermentation substrate, possibly after one or more additional treatments.

[0104] The liquid substrate, preferably gelatinized and preferably uncooled, containing saccharidases, preferably α-amylases, may undergo a dextrinization step, for example, after being introduced into a tank in which the substrate is maintained for a certain period. The duration of dextrinization is preferably 0.5 to 4 hours. This step allows the enzyme contained in the liquid substrate to continue hydrolyzing the starch. Advantageously, dextrinization is carried out until a dextrose equivalent (DE) of 4 to 14 is obtained. The DE is an indicator of starch hydrolysis. At a DE of 0, the starch is intact. At a DE of 100, the starch is completely converted to glucose. The method used to measure the DE is the Lane-Eynon method. The resulting substrate is then called "dextrinized."

[0105] The dextrinized substrate can be used as a fermentation substrate, possibly after one or more additional treatments.

[0106] The process according to the invention may, in particular, include a step of introducing at least one enzyme into the liquid substrate (in particular, into the starch fluid, the gelatinized substrate, or the dextrinized substrate), preferably selected from the group consisting of glucosidases. This step is called "saccharification" and enables the hydrolysis of dextrins into glucose. In this text, the term "saccharification" is used to designate any process of hydrolyzing dextrins into glucose, regardless of the degree of hydrolysis achieved; the saccharification step may also be called "pre-saccharification" when the hydrolysis is not complete or nearly complete.

[0107] Prior to the introduction of the enzymes, the liquid substrate can be introduced into a tank.

[0108] The enzyme(s) introduced are preferably chosen from the group consisting of α-1,4-glucosidases, amylo-α-1,6-glucosidases, and mixtures thereof. More preferably, at least two enzymes are introduced into the liquid substrate; even more preferably, at least one α-1,4-glucosidase and one amylo-α-1,6-glucosidase are introduced into the liquid substrate. The α-1,4-glucosidase hydrolyzes the α-(1,4) bonds involved in the linear glucose chains of dextrins; the amylo-α-1,6-glucosidase enzyme (also called the "debranching enzyme") hydrolyzes the bonds involved in the branching of the chains. Advantageously, the pH is adjusted to a value of 3.5 to 5.0, preferably 4.0 to 4.5. The temperature of the medium is preferably maintained between 50 and 70°C, preferably between 55 and 65°C. These conditions allow for optimal enzyme function. The quantity of enzymes introduced can be from 0.5 to 3.0 kg per tonne of assimilable carbohydrates.

[0109] Preferably, to carry out saccharification, the liquid substrate containing the glucosidases is kept in its container for a certain period of time. Preferably, the saccharification period is from 0.5 to 6 hours, preferably from 0.5 to 2 hours (in these embodiments, this step is more specifically referred to as "pre-saccharification").

[0110] In some embodiments, dextrinization and saccharification can be simultaneous (at least in part).

[0111] A glucose-enriched substrate is obtained, usable as a fermentation substrate, either as is or after further processing, such as purification, particularly filtration and / or demineralization. A "glucose-enriched medium" is defined as a medium in which the glucose concentration is higher than that of the substrate before saccharification.

[0112] Preferably, when the substrate undergoes a pre-saccharification step of 0.5 to 6 hours, it is not subjected to further purification. The hydrolyzed substrate can be used as a fermentation substrate.

[0113] Advantageously, the preparation of the fermentation substrate as described above generates a greenhouse gas emission of less than 100 kg of CO2 oil equivalent (CCheq) per tonne of plant material used, preferably less than 60 kg of CCheq per tonne of plant material used, more preferably less than 40 kg of CCheq per tonne of plant material used.

[0114] The hydrolyzed substrate advantageously comprises one or more of the following characteristics:

[0115] - a quantity of protein, relative to the total dry weight of the substrate, of 5 to 25% by weight, preferably 15 to 20% by weight, for example 5 to 10% by weight, or 10 to 15% by weight, or 15 to 20% by weight, or 20 to 25% by weight;

[0116] - an amount of assimilable carbohydrate, relative to the total dry weight of the substrate, of 40 to 80% by weight, preferably 60 to 75% by weight, for example 40 to 50% by weight, or 50 to 60% by weight, or 60 to 70% by weight, or 70 to 80% by weight;

[0117] - a quantity of fat (lipids), relative to the total dry weight of the substrate, of 0.5 to 5% by weight, preferably 0.5 to 3% by weight, for example 0.5 to 1% by weight, or 1 to 2% by weight, or 2 to 3% by weight, or 3 to 4% by weight, or 4 to 5% by weight;

[0118] - a quantity of fiber, relative to the total dry weight of the substrate, of 2 to 10% by weight, preferably 3 to 6% by weight, for example 2 to 4% by weight, or 4 to 6% by weight, or 6 to 8% by weight, or 8 to 10% by weight.

[0119] The amounts of protein, digestible carbohydrate, fat and fiber can be determined as indicated above.

[0120] The process according to the invention comprises the use of at least one "deviscosifying" enzyme, preferably at least two such enzymes. "Deviscosifying enzymes" are understood to be hydrolase enzymes capable of hydrolyzing water-insoluble fibers. Preferably, the deviscosifying enzymes according to the invention are selected from the group consisting of glucanases, preferably beta-glucanases, xylanases, galactosidases, preferably alpha-galactosidases, and combinations thereof. According to the invention, the deviscosifying enzymes can be used before and / or after the fermentation step.

[0121] Thus, according to one variant of the process, the liquid substrate can be contacted with at least one deviscosifying enzyme, preferably at least two deviscosifying enzymes. More preferably, the liquid substrate is contacted with at least two enzymes selected from the group consisting of glucanases, preferably beta-glucanases, xylanases, galactosidases, preferably alpha-galactosidases, and combinations thereof.For example, the liquid substrate may be contacted with at least one glucanase, preferably at least one beta-glucanase, and with at least one xylanase; or with at least one glucanase, preferably at least one beta-glucanase, and with at least one galactosidase, preferably one alpha-galactosidase; or with at least one xylanase and with at least one galactosidase, preferably one alpha-galactosidase; or with at least one glucanase, preferably at least one beta-glucanase, with one xylanase, and with at least one galactosidase, preferably one alpha-galactosidase. Particularly preferred, the liquid substrate is contacted with at least one glucanase, preferably at least one beta-glucanase, and with at least one xylanase, or with one glucanase, preferably at least one beta-glucanase, and with at least one galactosidase, preferably one alpha-galactosidase.The action of these enzymes is to hydrolyze water-insoluble fibers to make them water-soluble. The amount of devicolytic enzyme introduced into the liquid substrate can be from 0.2 to 4.0 kg per ton of dry matter (of the liquid substrate), preferably from 0.5 to 4.0 kg per ton of dry matter. Preferably, at least one glucanase is introduced in an amount of 0.5 to 2.0 kg per ton of dry matter and / or (preferably and) at least one xylanase in an amount of 0.5 to 2.0 kg per ton of dry matter. In an alternative, also preferred, embodiment, at least one glucanase may be introduced in an amount of 0.5 to 2.0 kg per ton of dry matter and / or (preferably and) at least one galactosidase in an amount of 0.1 to 1.0 kg per ton of dry matter.

[0122] In this embodiment, the introduction of the deviscosifying enzyme(s), or each of them independently, into the substrate can take place at any stage of the process according to the invention, from the formation of the liquid substrate up to the fermentation stage. Thus, the introduction of these enzymes can occur after one or more of the following stages: formation of the liquid substrate, hydrolysis, dextrinization, gelatinization, saccharification, introduction of amylases, and introduction of glucosidases. Preferably, the deviscosifying enzymes are introduced before the gelatinization stage, specifically after the formation of the liquid substrate (starch fluid) or after the introduction of the saccharidases (preferably α-amylases).

[0123] The process according to the invention includes a fermentation step of the liquid substrate (preferably the hydrolyzed substrate). More particularly, to carry out this fermentation step, the process includes contacting the liquid substrate with at least one microorganism. The microorganism is preferably selected from the group consisting of yeasts, bacteria, and combinations thereof. More preferably, the liquid substrate is contacted with at least one yeast, in particular at least one yeast of the genus Saccharomyces, for example, Saccharomyces cerevisiae and / or Saccharomyces bayanus. Microorganisms capable of fermenting glucose are known to those skilled in the art. Preferably, the microorganism, preferably the yeast, is added in an amount of 1 to 5 kg per ton of dry matter of the liquid substrate.

[0124] In some embodiments, the microorganism is brought into contact with a glucose-enriched substrate (i.e., one that has undergone the saccharification step) as described above, serving as a fermentation substrate. Preferably, the substrate is introduced into a tank, and the microorganism is then added to the tank. Preferably, the substrate has been cooled to a temperature of 20 to 40°C, more preferably 25 to 35°C, and even more preferably 26 to 32°C, prior to its contact with the microorganism. Preferably, the pH is maintained at a value of 3.5 to 5.0, and more preferably 4.0 to 4.5, during the fermentation step.

[0125] In other embodiments, the microorganism, preferably one or more yeasts, is brought into contact with a dextrinized substrate as described above, serving as a fermentation substrate. Preferably, the dextrinized substrate is pre-cooled to a temperature of 20 to 40°C, more preferably 25 to 35°C, and more preferably 26 to 32°C. Preferably, the substrate is brought into contact with the microorganism and with at least one enzyme, more preferably at least two enzymes, preferably selected from the group consisting of glucosidases. Particularly preferred, the substrate is brought into contact with the microorganism and with at least one α-1,4-glucosidase and one amylo-α-1,6-glucosidase. This step is called the "propagation step." Advantageously, the substrate is introduced into a vessel, and the microorganism and enzymes are added to the vessel.The enzyme quantities and pH are advantageously as described above in relation to the saccharification step. In these embodiments, dextrin hydrolysis and fermentation are partially carried out simultaneously.

[0126] In other embodiments, the microorganism, preferably one or more yeasts, is brought into contact with a gelatinized substrate as described above, serving as a fermentation substrate. Preferably, the gelatinized substrate is pre-cooled to a temperature of 20 to 40°C, more preferably 25 to 35°C, and more preferably 26 to 32°C. Preferably, the substrate is brought into contact with the microorganism and with at least one enzyme, preferably several enzymes, more preferably at least one enzyme selected from the saccharidases, preferably the α-amylases, and at least one enzyme, more preferably at least two enzymes, selected from the glucosidases. Most preferably, the substrate is brought into contact with the microorganism and with at least one α-amylase, one α-1,4-glucosidase, and one amylo-α-1,6-glucosidase.Advantageously, the substrate is introduced into a tank, and the microorganism and enzymes are added to the tank. The enzyme quantities and pH are advantageously as described above in relation to the dextrinization and saccharification steps. In these embodiments, starch hydrolysis and fermentation are partially carried out simultaneously. This has the advantage of limiting the residence time of the medium at high temperature, and thus limiting the risk of formation of undesirable molecules.

[0127] The process according to the invention may in particular include, after the formation of the liquid substrate, the following steps, in this order: introduction of saccharidases (preferably α-amylases), introduction of deviscosifying enzymes, gelatinization, dextrinization, introduction of glucosidases, saccharification, introduction of microorganisms and fermentation; or the following steps, in this order: introduction of saccharidases (preferably α-amylases), introduction of deviscosifying enzymes, gelatinization, introduction of glucosidases, simultaneous dextrinization and saccharification, introduction of microorganisms and fermentation; or the following steps, in this order: introduction of deviscosifying enzymes, gelatinization, introduction of saccharidases (preferably α-amylases) and glucosidases, introduction of microorganisms and fermentation.Alternatively, the process may include the above steps, in the order indicated, but without the step of introducing the deviscosifying enzymes, the introduction of the deviscosifying enzymes taking place after fermentation, and more specifically after distillation, as described below.

[0128] Contacting the fermentation substrate with the microorganism(s) advantageously results in obtaining a fermentation product. Preferably, the fermentation product comprises, or consists of, at least one alcohol, and preferably comprises, or is, ethanol.

[0129] Preferably, fermentation is carried out for a period of 36 to 70 hours, preferably 40 to 60 hours. Advantageously, at the end of fermentation, all the simple sugars (glucose and maltose) have been converted into alcohol and CO2. Advantageously, the alcohol content obtained is between 8 and 17°, preferably between 10 and 15° (corresponding to an alcohol content of 8 to 17% (v / v), preferably 10 to 15% (v / v)).

[0130] At the end of the fermentation process, a fermented medium is obtained.

[0131] The process according to the invention includes a distillation step of the fermented medium. The distillation can be carried out using any suitable distillation apparatus. In particular, a distillation column, more especially a column containing metal Raschig rings, can be advantageously used. The column is preferably fitted with a condenser. Preferably, an outlet pipe equipped with a diaphragm of variable diameter connects the column and the condenser. The vapors passing beyond the diaphragm are then condensed by the condenser.

[0132] The distillation of a fermented medium to recover the alcohols produced during fermentation is well known to those skilled in the art. The fermented medium is heated to the boiling point of the fermentation product, which becomes gaseous and is then recovered by condensation. Preferably, the fermented medium is heated to a temperature of 80 to 100°C. An example of the distillation process is as follows. Upon boiling, the alcohol vapors reach the diaphragm, which is initially closed. The vapors are then condensed by the upper condenser and fall back down the column. During this phase, there is a liquid / vapor exchange along the entire height of the column containing the Raschig rings, which allows for the purification of the alcohol vapors at the top of the distillation column. When the liquid / vapor exchange is balanced, the temperature of the alcohol vapors at the diaphragm is approximately 78°C.The diaphragm is then opened to a diameter that allows the temperature of the alcohol vapors to be maintained at approximately 78°C.

[0133] At the end of the distillation, a distillate is collected comprising the fermentation product, preferably alcohol, more preferably ethanol, and a distillation residue (also called vinasse).

[0134] Preferably, distillation is carried out until all, or almost all, of the alcohol has been distilled, i.e., the degree of alcohol in the distillation residue is less than or equal to 2°, preferably less than or equal to 1°, more preferably about 0°.

[0135] When deviscosifying enzymes were added to the medium before fermentation, the process according to the invention makes it possible to obtain a distillation residue containing a small amount of water-insoluble fiber.

[0136] When no deviscosifying enzyme has been introduced into the liquid substrate prior to fermentation, the process includes contacting the distillation residue with one or more deviscosifying enzymes. More specifically, the distillation residue is contacted with at least one, preferably at least two, enzyme(s) selected from the group consisting of glucanases, preferably beta-glucanases, xylanases, galactosidases, preferably alpha-galactosidases, and combinations thereof. The information described above regarding the introduction of deviscosifying enzymes into the liquid substrate (in particular, the number and type of enzymes introduced, and the quantities of enzymes introduced) can be applied similarly to the introduction of deviscosifying enzymes into the distillation residue.Introducing deviscosifying enzymes into the distillation residue rather than before fermentation improves alcohol production as a fermentation product. Preferably, the pH of the distillation residue is adjusted to a value of 3.5 to 5.0, preferably 4.0 to 4.5.

[0137] In some embodiments, deviscosifying enzymes can be introduced into the liquid substrate before fermentation and into the distillation residue.

[0138] The process according to the invention then includes a solid-liquid separation step, carried out on the distillation residue (where applicable, after the introduction of deviscosifying enzymes). Preferably, the solid-liquid separation is selected from separations by centrifugation, separations by filtration, such as membrane filtration and / or rotary drum filtration, separations by decantation, and combinations thereof. The solid-liquid separation may comprise one or more of the aforementioned separation steps. The solid-liquid separation results in a solid fraction and a liquid fraction. The water-insoluble compounds are thus separated from the water-soluble compounds contained in the liquid fraction.In particular, insoluble fibers were hydrolyzed, notably during fermentation or in the distillation residue, using deviscosifying enzymes such as glucanases, xylanases and galactosidases, transforming them into soluble compounds that will be transferred into the liquid phase and thus removed during solid-liquid separation.

[0139] The solid fraction is collected. The solid fraction preferably comprises a dry matter content of at least 20% by weight, and preferably at least 25% by weight. The solid fraction may be subjected to a drying step. The solid fraction, possibly dried, corresponds to a protein isolate.

[0140] Advantageously, the protein isolate produced by the process according to the invention comprises:

[0141] - a quantity of protein, relative to the total dry weight of the isolate, of 70 to 95% by weight, preferably 75 to 95% by weight, preferably still 75 to 90% by weight, more preferably 75 to 85% by weight, even more preferably 78 to 85% by weight, for example 70 to 75% by weight, or 75 to 80% by weight, or 80 to 85% by weight, or 85 to 90% by weight, or 90 to 95% by weight; and / or

[0142] - an amount of digestible carbohydrate, relative to the total dry weight of the isolate, of 1 to 15% by weight, preferably 1 to 10% by weight, preferably 2 to 8% by weight, more preferably 3 to 7% by weight, even more preferably 4 to 6% by weight, for example 1 to 3% by weight, or 3 to 6% by weight, or 6 to 8% by weight, or 8 to 10% by weight, or 10 to 13% by weight, or 13 to 15% by weight; and / or

[0143] - a quantity of fat (lipids), relative to the total dry weight of the isolate, of 0.5 to 5% by weight, preferably 0.5 to 4.5% by weight, more preferably 0.5 to 3.5% by weight, for example 0.5 to 2% by weight, or 2 to 3% by weight, or 3 to 4% by weight, or 4 to 5% by weight; and / or

[0144] - a quantity of fiber, relative to the total dry weight of the isolate, of 2 to 12% by weight, preferably 2 to 10% by weight, such as 3 to 6% by weight, for example 2 to 4% by weight, or 4 to 6% by weight, or 6 to 8% by weight, or 8 to 10% by weight, or 10 to 12% by weight.

[0145] The invention also relates to a protein isolate obtained by, or capable of being obtained by, a preparation process as described above. The protein isolate may be as described above.

[0146] Examples

[0147] The following examples illustrate the invention without limiting it.

[0148] In the examples, the following analysis and measurement methods were used:

[0149] - Measurement of the quantity of proteins: Kjeldahl method using a FOSS titrator according to ISO 5983-2;

[0150] - Measurement of the quantity of lipids: AOAC method 922.06;

[0151] - Quantity of assimilable carbohydrates: determined by calculating the difference between the total weight of the product and the sum of the weights of proteins, fats, total fibers, water and ash;

[0152] - Measurement of the quantity of ash: method according to the standard NF EN ISO 2171;

[0153] - Water content (or "moisture"): obtained by the difference between the initial total weight of the sample and the dry extract measured according to the ISO 6731 method;

[0154] - Measurement of the quantity of total fibers: according to the AOAC 2011.25 method;

[0155] - Measuring the alcohol content: using a densimeter.

[0156] Example 1: Production of a protein isolate from pea flour

[0157] 300 kg of peas are hulled using a STOLZ PEDT 600 roller mill (roller gap of 4.5 mm) and 30 kg of hulls are separated from the kernels using a STOLZ SNST 550 sieve equipped with a 5 mm sieve and a 2 mm sieve, and a STOLZ SEPAIR 400 air separator (with an air flow rate of 2600 m 3 / h).

[0158] 270 kg of almonds are ground in an Alpine-Hosokawa ZPS 500 mill operating at 2650 rpm to obtain micronized pea flour with a volume median particle diameter (D50) of 30 µm or less. This flour is then fed into an air separator using two successive separators: the first Alpine-Hosokawa ZPS 500 separator operates at 1600 rpm with an air flow rate of 5300 m³ / h. 3 / h, and a second Alpine-Hosokawa ATP 315 selector operating at a rotational speed of 3400 rpm and with an airflow rate of 3600 m 3 / h, in order to separate the protein-enriched fraction, weighing 70 kg, from the starch-enriched fraction, weighing 200 kg. During the aerodynamic separation, the lighter particles, with a D50 of 1 to 5 pm, are carried to the top of the cyclone, while the heavier particles, with a D50 of 10 to 30 pm, are carried to the bottom.

[0159] 200 kg of the starch-enriched fraction are mixed with 600 liters of potable water in an ISTILL 1000 reactor with a capacity of 1000 liters. The reactor is equipped with a distillation column, an electric heating element, and a radiator supplied with cold water for cooling the medium.

[0160] The composition of the starch-enriched fraction is shown in Table 1 below (as mass percentage).

[0161] Table 1 ]

[0162] The pH of the mixture is adjusted to 4.5 using sulfuric acid. In order to solubilize the fibers by hydrolysis, a beta-glucanase (Optimash® TBG marketed by IFF) and a xylanase (Viscamyl™ flow marketed by IFF) are added in quantities of 0.6 g / kg of dry matter of the mixture for the beta-glucanase and 0.6 g / kg of dry matter of the mixture for the xylanase.

[0163] The mixture is heated to 65°C using an electric heating element and maintained at this temperature for 30 minutes. The mixture is then cooled to 32°C. Alpha-amylase (LPHERA® alpha-amylase, marketed by NOVOZYMES) is introduced into the medium at a concentration of 2.7 g / kg of digestible carbohydrates. Simultaneously, a glucosidase mixture (Optidex® mixture, marketed by IFF, containing a blend of α-1,4-glucosidases and amylo-α-1,6-glucosidase) is introduced into the medium at a concentration of 2.7 g / kg of digestible carbohydrates.

[0164] A Saccharomyces bayanus yeast is introduced at a concentration of 3.5 g / kg of dry matter in the mixture. 0.15 mL of vegetable oil-based antifoam is added per liter of reaction medium.

[0165] The medium is then cooled until it reaches a temperature of 25°C.

[0166] Fermentation is carried out by maintaining the medium at 25°C for 60 hours, adjusting the pH with caustic soda to maintain it between 4.0 and 4.5. The reaction medium is gently stirred to promote anaerobic yeast activity. The alcohol content (specifically, ethanol) obtained after 60 hours of fermentation is 10.2% v / v. The medium is then heated to its boiling point to distill all the alcohol produced during fermentation.

[0167] After distillation, the distillation residue, or "vinasse," is centrifuged at 4500 rpm. The insoluble portion (also called the solid fraction) is recovered after removing the supernatant and is introduced into a vacuum drum dryer. The drying temperature is 40°C.

[0168] The composition of the product obtained is shown in Table 2 below, as a percentage on a dry matter basis.

[0169] [Table 2]

[0170] It is observed that a protein isolate with a very high protein content has been obtained.

[0171] Example 2: Production of a protein isolate according to other parameters

[0172] 130 kg of starch-enriched fraction obtained by dehulling, micronization, and air separation as described in Example 1 are mixed with 500 liters of potable water in an ISTILL 1000 reactor with a capacity of 1000 liters. The reactor is equipped with a distillation column, an electric heating element, and a cold-water radiator for cooling. The starch-enriched fraction is as described in Example 1, and its composition is that indicated in Table 1 above.

[0173] The pH of the mixture is adjusted to 4.5 using sulfuric acid before the addition of acid alpha-amylase (LPHERA® alpha-amylase, marketed by NOVOZYMES) at a concentration of 3.9 g / kg of digestible carbohydrates. To solubilize (by hydrolysis) the fiber, beta-glucanase and xylanase are added at concentrations of 1.1 g / kg of dry matter for beta-glucanase and 1.1 g / kg of dry matter for xylanase.

[0174] The mixture is heated to a temperature of 65°C and maintained at this temperature for 30 minutes. The mixture is then cooled to 60°C. A glucosidase mixture (Optidex® mixture marketed by IFF) is then introduced into the medium at a concentration of 1.1 g / kg of assimilable glucose, and the mixture is held at temperature for one hour.

[0175] Following this residence time, the medium is cooled to 30°C. A Saccharomyces cerevisiae yeast is introduced at a concentration of 1.4 g / kg of dry matter in the mixture. 20 mL of a vegetable oil-based antifoaming agent is added to the medium.

[0176] Fermentation is carried out by maintaining the medium at 30°C for 60 hours, adjusting the pH with caustic soda to maintain it between 4.0 and 4.5. The alcohol content (specifically, ethanol) obtained after 60 hours of fermentation is 10.9% v / v. The medium is then heated to its boiling point to distill all the alcohol produced during fermentation.

[0177] After distillation, the distillation residue or "vinasse" has the following composition:

[0178] Table 3]

[0179] The distillation residue is then centrifuged at 4500 rpm. The insoluble portion (also called the solid fraction) is recovered after removing the supernatant and introduced into a vacuum drum dryer. The drying temperature is 40°C.

[0180] The product composition is shown in Table 3 below as a percentage on a dry matter basis.

[0181] Table 4]

[0182] The molecular weight distribution profile of the product's proteins was analyzed by permeametry and is shown in Table 4 below (as a mass percentage). Approximately 90% by weight of the proteins have a molecular weight greater than 1000 kDa, and 68% by weight even have a molecular weight greater than 5000 kDa. The degree of hydrolysis is therefore low, indicating minimal hydrolysis of the proteins into peptides or amino acids.

[0183] Table 5]

[0184] Example 3: Production of a protein isolate by using deviscosifying enzymes in the distillation residue.

[0185] 130 kg of starch-enriched fraction obtained by dehulling, micronization, and air separation as described in Example 1 are mixed with 500 liters of potable water in an ISTILL 1000 reactor with a capacity of 1000 liters. The reactor is equipped with a distillation column, an electric heating element, and a cold-water radiator for cooling the medium.

[0186] The starch-enriched fraction is as described in Example 1 and its composition is that shown in Table 1 above.

[0187] The pH of the mixture is adjusted to 4.5 using sulfuric acid before the addition of acid alpha-amylase (LPHERA® alpha-amylase, marketed by NOVOZYMES) at a concentration of 3.9 g / kg of digestible carbohydrates. Unlike the process described in Example 2, no deviscosifying enzyme is added to the medium at this stage of the process.

[0188] The mixture is heated to a temperature of 65°C and maintained at this temperature for 30 minutes. The mixture is then cooled to 60°C. A glucosidase mixture (Optidex® mixture marketed by IFF) is then introduced into the medium at a concentration of 1.1 g / kg of assimilable glucose, and the mixture is held at temperature for one hour.

[0189] Following this residence time, the medium is cooled to 30°C. A Saccharomyces cerevisiae yeast is introduced at a concentration of 1.4 g / kg of dry matter in the mixture. 20 mL of a vegetable oil-based antifoaming agent is added to the medium.

[0190] Fermentation is carried out by maintaining the medium at 30°C for 60 hours, adjusting the pH with caustic soda to maintain it between 4.0 and 4.5. The alcohol content (specifically, ethanol) obtained after 60 hours of fermentation is 10.2% v / v. The medium is then heated to its boiling point to distill all the alcohol produced during fermentation.

[0191] After distillation, the distillation residue or "vinasse" has the following composition on a dry matter basis:

[0192] Table 6]

[0193] 20 litres of vinasse having a dry matter content of 10.5% by weight are brought to 50°C with the introduction of a beta-glucanase (VERTERA RELEASE, from the supplier NOVONESIS) in a quantity of 1 g / kg of dry matter and an alpha-galactosidase (VERTERA MELLO, from NOVONESIS) in a quantity of 0.2 g / kg of dry matter.

[0194] The pH of the medium is adjusted to 4.2.

[0195] The distillation residue is then centrifuged at 4500 rpm. The insoluble portion (also called the solid fraction) is recovered after removing the supernatant and introduced into a vacuum drum dryer. The drying temperature is 40°C.

[0196] The product composition is shown in Table 7 below as a percentage on a dry matter basis.

[0197] Table 7]

Claims

Demands 1. A process for preparing a protein isolate, comprising the following steps: - the supply of at least one seed comprising starch and proteins; - the micronization of said at least one seed, so as to obtain a micronized fraction; - the treatment of the micronized fraction by air separation, in order to collect a fraction enriched in starch; - mixing the starch-enriched fraction with an aqueous liquid, so as to form a liquid substrate; - the hydrolysis of starch in the liquid substrate, so as to obtain a hydrolyzed substrate; - the fermentation of the hydrolyzed substrate, in order to obtain a fermented medium; - the distillation of the fermented medium, so as to collect a distillate and a distillation residue; - the treatment of the distillation residue by solid-liquid separation, so as to obtain a liquid fraction and a solid fraction; and - the collection of the solid fraction; the process further comprising a step of bringing the liquid substrate or the hydrolyzed substrate and / or the distillation residue into contact with at least one enzyme selected from the group consisting of xylanases, glucanases, galactosidases and combinations thereof.

2. A method according to claim 1, wherein at least one seed is selected from the group consisting of legume seeds, preferably selected from the group consisting of beans, peas, broad beans, lentils, chickpeas, lupins, field beans and mixtures thereof; cereal seeds, preferably selected from the group consisting of oats, barley, wheat, maize and mixtures thereof; and combinations thereof.

3. A process according to claim 1 or 2, wherein the hydrolysis of starch comprises contacting the liquid substrate with at least one enzyme selected from the group consisting of saccharidases, preferably at least one α-amylase.

4. A process according to any one of claims 1 to 3, wherein the hydrolysis of the starch includes the gelatinization of the liquid substrate, so as to obtain a gelatinized substrate, preferably by heating said liquid substrate, more preferably the heating being carried out by mixing steam with the liquid substrate.

5. A method according to any one of claims 1 to 4, wherein at least one seed comprises a husk, and wherein the method comprises a seed dehulling step prior to micronization.

6. A process according to any one of claims 1 to 5, wherein the hydrolysis of starch comprises a step of contacting the liquid substrate with at least one enzyme selected from the group consisting of glucosidases, preferably with at least two enzymes selected from the group consisting of glucosidases, more preferably with at least one α-1,4-glucosidase and one amylo-α-1,6-glucosidase.

7. A process according to any one of claims 1 to 6, wherein the liquid substrate or the hydrolyzed substrate and / or the distillation residue are contacted with at least one xylanase and at least one glucanase, preferably at least one beta-glucanase; or with at least one glucanase, preferably at least one beta-glucanase, and with at least one galactosidase, preferably at least one alpha-galactosidase.

8. A method according to any one of claims 1 to 7, wherein the aerodynamic separation of the micronized fraction is carried out by means of a cyclone with a selector.

9. A method according to any one of claims 1 to 8, wherein the solid-liquid separation is selected from separations by centrifugation, filtration, decantation and combinations thereof.

10. A process according to any one of claims 1 to 9, comprising, for carrying out the fermentation, bringing the liquid substrate or the hydrolyzed substrate into contact with at least one microorganism, preferably chosen from the group consisting of yeasts, bacteria and combinations thereof, more preferably at least one microorganism is at least one yeast.

11. A process according to any one of claims 1 to 10, comprising a step of drying the solid fraction.

12. A method according to any one of claims 1 to 11, wherein the protein isolate comprises at least 70% by weight, preferably at least 75% by weight, of protein, relative to the total dry weight of the protein isolate.

13. A process according to any one of claims 1 to 12, wherein a fermentation product is produced during fermentation, said fermentation product being selected from alcohols, and preferably being ethanol.

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