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: supplying a seed; micronizing the seed to obtain a micronized fraction; treating the micronized fraction by air 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; contacting the liquid substrate with at least one enzyme selected from xylanases, glucanases, and combinations thereof; 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 by solid-liquid separation to obtain a liquid fraction and a solid fraction; collecting the solid fraction. No figure.

Description

Title of the invention: Method for preparing a plant protein isolate by fermentation. Field of the invention

[0001] The present invention relates to a method for producing a plant protein isolate from starch-containing plants, such as from legumes or cereals. Technical background

[0002] Global demand for protein is growing rapidly, linked to population growth on all continents and greater prosperity, particularly in Asia and Africa. However, recent climate crises, which are worsening year after year, are forcing governments to implement proactive environmental policies to reduce greenhouse gas emissions.

[0003] Proteins are currently derived overwhelmingly from animal sources. Cattle, sheep, and poultry farming generate significant water consumption and high greenhouse gas emissions. Plant-based protein production, on the other hand, has a considerably less negative environmental impact.

[0004] The transition from animal to plant proteins is not, however, simple. Products derived from the animal world are preferred due to their taste qualities, which are 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 therefore these characteristics specific to the plant world that have favored the development of processes for fractionating plant products.

[0005] Among plants containing proteins, three classes of plants are most used: cereals (especially wheat and corn) in the majority, followed by oilseeds (notably soybeans and sunflowers) and then legumes.

[0006] Historical methods of extracting proteins from plants are so-called wet processes, which differ according to the class of plant.

[0007] In the case of wheat, which contains vital gluten as a source of protein, extraction begins with a series of millings of the grains 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. The mixture is then sent to a three-phase decanter, from which three fluids are separated: 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 per tonne of wheat. Therefore, there is a double negative environmental impact: • The first is related to the use of large 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 approximately 2 million m3 of water per year. • The second is linked to the intensive use of fertilizers for wheat cultivation, its transport, and all the energy required for the gluten extraction process, which includes the evaporation of water consumed upstream. The environmental impact in greenhouse gases estimated by the CarbonCloud platform is 2,300 kg CO2eq / t of wheat gluten produced.

[0008] The process of extracting gluten from maize begins with a soaking step to facilitate milling. Subsequent steps use various separation methods (filter screens, centrifuges, hydrocyclones) and result in three products: gluten, oil-rich germ, and starch. The gluten is concentrated using a decanter or a rotary vacuum filter and then dried. As with wheat, this process also has a dual negative environmental impact, linked to: • Water consumption is estimated at 1.4 m3 per tonne of corn. For a medium-sized starch factory that grinds 1,500 tonnes of corn per day, this represents a withdrawal of approximately 700,000 m3 of water per year. • the use here also of fertilizers and a need for energy necessary for the evaporation of water which results in a greenhouse gas impact of 1,700 kg CO2eq per tonne of corn gluten produced (source: CarbonCloud).

[0009] In the case of oilseeds such as soybeans and sunflowers, industrial-scale fractionation is carried out beforehand by extracting the fats with 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 dried. The environmental impact is therefore mainly related to plant 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 CO2eq per ton of soybeans used and, for sunflowers, at between 2,000 and 3,000 kg CO2eq per ton of sunflowers used.

[0010] With regard to legumes, the protein extraction process includes firstly a step of grinding the seed to obtain a flour.

[0011] According to a first protein extraction method, the ground seeds are suspended in an alkaline solution with a pH close to 9. Proteins of the globulin and albumin family (which typically represent between 75 and 85% by weight of the total proteins) 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 carried out to recover the solid phase. After resuspension, the pH can be adjusted to neutral before drying the proteins.With this process, proteins of the glutelin family are not extracted because the pH that would need to be applied for their extraction would cause loss of functionality of the albumins and globulins.

[0012] Another method for processing legumes is based on the use of filtration membranes. In this process, the legume seed is ground to obtain a flour. This flour is 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 the starch and fibers is separated from the proteins solubilized in the solvent. The supernatant containing the proteins is filtered through organic membranes with a porosity of 10 kDa. Proteins with a high molecular weight are retained in the recirculation loop and recovered in the retentate. The smaller molecules that pass through the membrane are collected in the permeate. The retentate is then subjected to a diafiltration step, which consists of filtering the diluted proteins again with demineralized water.This step aims to "wash" the proteins in order to minimize the presence of salts and amino acids. The proteins are then concentrated to approximately 30% by weight of dry matter and dried using a spray dryer.

[0013] The techniques used for extracting legume proteins 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 of CO2eq per tonne of protein isolate produced (source: CarbonCloud). However, the most significant impact is caused by the high consumption of water and chemicals, and consequently the volume of wastewater requiring treatment.

[0014] Other methods for producing protein fractions have been described.

[0015] 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. This process includes a grinding stage for unhulled peas, a sieving stage, and an air separation stage for the ground peas, resulting in a fraction of larger particles and a fraction of finer particles, enriched in protein. The resulting protein concentrates contain 51 to 55% protein (on a dry matter basis).

[0016] US patent 9,476,068 discloses a process for preparing a co-product from a 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.

[0017] 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 comprise up to 58% by weight of protein.

[0018] These processes allow the obtaining of fractions comprising an insufficient protein content on a dry matter basis.

[0019] There is therefore a real need to develop a process for obtaining a protein isolate from plant material comprising a higher protein content and having a lower greenhouse gas emission. Summary of the invention

[0020] The invention relates primarily to a method for preparing a protein isolate, comprising the following steps: • the provision of at least one seed comprising starch and protein; • the micronization of said at least one seed, so as to obtain a micronized fraction; • the treatment of the micronized fraction by air separation, so as 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; • bringing the liquid substrate into contact with at least one enzyme chosen from the group consisting of xylanases, glucanases and combinations thereof; • 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 a solid-liquid separation, so as to obtain a liquid fraction and a solid fraction; • the collection of the solid fraction.

[0021] 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, broad beans and mixtures thereof; cereal seeds, preferably chosen from the group consisting of oats, barley, wheat, maize and mixtures thereof; and combinations thereof.

[0022] In embodiments, the hydrolysis of starch includes bringing the liquid substrate into contact with at least one enzyme selected from the group consisting of saccharidases, preferably at least one α-amylase.

[0023] In 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.

[0024] In embodiments, at least one seed comprises a film, and the process includes a step of de-filming the seed, prior to micronization.

[0025] 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 of at least two enzymes chosen from the group consisting of glucosidases, more preferably of at least one α-1,4-glucosidase and one amylo-α,6-glucosidase.

[0026] In embodiments, the liquid substrate is brought into contact with at least one xylanase and at least one glucanase, preferably at least one beta-glucanase.

[0027] In embodiments, the aerodynamic separation of the micronized fraction is carried out by means of a cyclone with selector.

[0028] In embodiments, the solid-liquid separation is chosen from separations by centrifugation, by filtration, by decantation and combinations thereof.

[0029] In embodiments, fermentation includes bringing the liquid substrate or 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.

[0030] In embodiments, the process includes a step of drying the solid fraction.

[0031] In embodiments, the protein isolate comprises at least 75% by weight of protein, relative to the total dry weight of the protein isolate.

[0032] In embodiments, a fermentation product is produced during fermentation, said fermentation product being chosen from alcohols, and preferably being ethanol.

[0033] The present invention addresses the need expressed above. More particularly, it provides a process for preparing a protein isolate from plant material that is more environmentally friendly, and more specifically, has low greenhouse gas emissions, and is easily implemented on an industrial scale, while still allowing for a high protein content. Furthermore, the process according to the invention enables the production of other products of interest such as ethanol or other organic compounds.

[0034] This is achieved through the implementation of a particular sequence of steps including in particular micronization of the plant material, aeraulic separation, hydrolysis by means of deviscosifying enzymes (in particular xylanases and / or glucanases), fermentation, distillation and solid-liquid separation of the distillation residue.

[0035] Furthermore, in embodiments where the plant material is a legume, the process according to the invention makes it possible to further reduce greenhouse gas emissions. Indeed, legumes have the ability to use atmospheric nitrogen for their growth, via organisms Symbiotic organisms 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. Detailed description

[0036] The invention is now described in more detail and in a non-limiting manner in the following description.

[0037] Unless otherwise indicated, all percentages are mass percentages.

[0038] 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.

[0039] 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 75% by weight of protein, more preferably at least 78% by weight, relative to the total dry weight of the fraction.

[0040] Preferably, the plant material further comprises fibers. "Fiber" means 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 further comprise lipids (or fats).

[0041] Preferably, the plant material is derived from at least one legume, at least one cereal, or combinations thereof.

[0042] The term "legume" refers to plants of the Fabaceae family. Legume seeds contain, in particular, 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 a protein content of between 20 and 30% by weight on a dry matter basis) and of having a low fat content.

[0043] All legumes are suitable for the invention. Examples of legumes that can be used in the invention include, in particular, beans, peas, broad beans, lentils, chickpeas, lupins, broad beans and mixtures thereof.

[0044] 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.

[0045] Advantageously, the plant material according to the invention comprises at least one seed. More advantageously, the plant material is at least one seed.

[0046] Generally, seeds comprise at least one kernel surrounded by a hull (or husk). However, in the present text, the term "seed" may generally refer to the whole (complete) seed as well as any part of the seed (for example, the kernel), unless otherwise specified.

[0047] The seeds used in the invention as starting plant material preferably comprise a skin and a kernel.

[0048] Even more advantageously, the plant material comprises, or is, at least one legume seed. The use of legumes as a raw material is advantageous 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 bacteria of the genus Rhizobium via the formation of nodules, thus providing the plant with the nitrogen necessary for its growth. The ability of legumes to fix atmospheric nitrogen makes it possible to avoid the use of 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, which is 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 CO2eq per tonne of legume.When legume crops are combined 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 CO2eq per tonne of legume: the legume crop therefore has a net balance of almost neutral 11 kg CO2eq per tonne of legume in this case.

[0049] Preferably, the plant material comprises, or is, a pea seed.

[0050] Plant material, in particular the seed, may include a husk. Advantageously, the process according to the invention includes a step of removing the seed coat (or dehulling). Indeed, the seed coat is composed mainly of insoluble fibers that are not consumed by fermentation microorganisms. Furthermore, since the majority of seed contaminants are found in the seed coat, its removal reduces the risk of contamination of the prepared isolate.

[0051] Preferably the peeling is a mechanical peeling, carried out for example by abrasion, compression, impact, shearing or any other appropriate mechanical action.

[0052] In some embodiments, the dehulling is carried out by grinding the seed and then separating the particles obtained according to their size and / or density.

[0053] For grinding, any suitable type of grinder can be used, in particular any grinder using one of the mechanical forces mentioned above. In particular, a roller mill (also called a cylinder mill) using compression 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 kernel pieces is obtained.

[0054] The step of separating the particles obtained can in particular be carried out by sieving, by air separation, or by a combination of these two techniques.

[0055] The separation step is preferably carried out by sieving followed by air separation.

[0056] Thus, the particles obtained after grinding can undergo one or more sieving steps. In particular, the particles can be separated by passing them through a sieve with a suitable mesh size, allowing the separation of larger skin fragments and kernel pieces from smaller skin fragments and kernel pieces (resulting in two particle fractions of different sizes). In particular, at least one sieving can be carried out 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, so as 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.

[0057] The particles containing the film fragments and the kernel pieces, preferably at least one of the fractions obtained after sieving, 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 (preferably air) is preferably from 1500 to 3500 m³ / h, more preferably from 2000 to 3000 m³ / h.

[0058] Optionally, when the plant material is a cereal seed, the process may include a step of dehulling (or removing awns) the seed, carried out by example by abrasion, more particularly using a deburring tool, this step preferably being carried out before peeling.

[0059] The plant material, optionally dehulled, optionally in the form of pieces, is subjected to a micronization step and a micronized fraction is collected. Advantageously, the micronization is a dry micronization.

[0060] By "micronization" is meant a grinding process that produces particles with a volume median diameter of less than 100 pm, preferably less than 50 pm, and even more preferably less than 30 pm. The volume median diameter (D50) of the particles can be measured according to standard NF ISO 13320-1.

[0061] 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.

[0062] Micronization is most preferably carried out at room temperature (i.e. between 15 and 30 °C).

[0063] Micronization is preferably carried out at a mill speed of 2000 to 3000 rpm, preferably still 2500 to 3000 rpm.

[0064] Micronization has the additional advantage of being low emitter of greenhouse gases, preferably being carried out at ambient temperature and by dry process.

[0065] The process according to the invention includes a step of purifying the micronized fraction. Advantageously, this purification includes a step of separating the starch granules from the proteins.

[0066] 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 fraction subjected to purification. 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 fraction subjected to purification.

[0067] Given the difference in size and density between proteins and starch granules (D50 of approximately 1 to 5 µm for proteins and approximately 10 to 30 µm for starch granules), separation based on a difference in particle size, density, or weight can advantageously be used. Most preferably, the separation is an air separation. "Air separation" means any separation technology using a jet of gas (preferably air) that carries away at least some of the particles to be separated. More preferably, the separation This is a cyclonic separation. It can be carried out using a cyclone, advantageously combined with at least one selector, and more advantageously with at least two selectors. A "selector" is defined as any variable-speed rotating element equipped with a slotted cylinder installed in a part (preferably the upper part) of a cyclonic separator. This equipment increases the separation efficiency of particles according to their density. The use of an air separation device allows the recovery of the lightest particles, carried by the gas flow (preferably air), at one end of the device (protein-enriched fraction), while the heavier particles are collected at the other end (starch-enriched fraction).Preferably, the lighter particles are carried upwards by a vertical gas flow in the apparatus, and the heavier particles, not carried upwards by the gas flow, fall downwards in the vertical gas flow.

[0068] The air separation is preferably carried out using a selector operating at a rotational speed between 1000 and 3000 rpm, more 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 / h, more preferably between 5000 and 5500 m³ / h. Preferably, the selector is followed by a second selector operating preferably at a rotational speed between 2500 and 4500 rpm, more preferably between 3000 and 3500 rpm. The flow rate of the gas flow (preferably air) through the second selector is preferably between 2500 and 5000 m³ / h, and more preferably between 3500 and 4000 m³ / h. When air 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.

[0069] 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, this tendency being even more pronounced with regard to fiber. The starch-enriched fraction is thus purified of some of these components. This is achieved by carrying out the micronization step prior to air separation, which promotes the transfer of fiber and ash to the protein-enriched fraction. Indeed, using coarser grinding instead of micronization would lead to a very imperfect separation of proteins, starch, fiber, and ash during air separation, and therefore to obtaining a starch-enriched fraction that is much less purified of fiber and ash.

[0070] The advantage of this method of obtaining the fractions, by micronization and then air separation, is that it is low in greenhouse gas emissions and 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 includes fibers, fats, and salts) generally cannot exceed 60 to 65% on a dry matter basis.

[0071] Thus, the process according to the invention has the advantage, compared to a process for producing a protein fraction based on the recovery of the protein-enriched fraction, as described in the article by Pelgrom et al. mentioned above, of allowing a higher protein content to be obtained. 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.

[0072] 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 at the end of the micronization step.

[0073] 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, more preferably 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(λ-4) or α(λ-6) bonds, including starch and dextrins.

[0074] 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.

[0075] Preferably, the starch-enriched fraction comprises a fiber content of 10% or less by weight, preferably 0.5 to 10% by weight, and more preferably 1 to 6% by weight, relative to the total dry matter weight of the fraction; in particular the fraction may comprise an amount of fibre 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.

[0076] 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, more preferably 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.

[0077] The quantities of protein, digestible carbohydrate, fat and fiber can be determined as indicated below in the Examples section.

[0078] Advantageously, the starch-enriched fraction is then mixed with an aqueous liquid to form a liquid substrate, more particularly a fluid comprising starch or starch fluid. The aqueous liquid is preferably water. The starch fluid is preferably in the form of a dispersion, more preferably a starch milk. "Starch milk" means a suspension of starch in water (said suspension may include other components, whether or not they are solubilized in water).

[0079] In this text, "liquid substrate" means 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).

[0080] Preferably, the liquid substrate (the starch fluid, preferably the 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.

[0081] 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.

[0082] 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, particularly 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.

[0083] 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 in order to release the starch molecules into the liquid, thus allowing the action of enzymes (which otherwise do not have access to the starch molecules enclosed in the granules). The bursting of the starch granules is preferably achieved by heating the liquid substrate (preferably starch fluid) to a temperature that allows the liquid to be introduced into the granule, thereby causing the granule to swell and then burst. A gelatinized substrate is then obtained.

[0084] Gelatinization can be carried out by any type of heating.

[0085] 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.

[0086] Preferably, the liquid substrate (preferably, the starch fluid) is heated (preferably by mixing with steam) to a temperature (referred to in this text as the gelatinization temperature) of 50 to 130 °C, preferably 50 to 100 °C.

[0087] Particularly advantageously, gelatinization is carried out by mixing a stream of water vapor with a stream of the liquid substrate (preferably starch fluid). By "stream" is meant a fluid (gas or liquid) in motion.

[0088] 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 partly simultaneously with the discharge of the mixer of said mixture.

[0089] Mixing steam with the liquid substrate (preferably starch fluid) in a continuous flow allows for a very rapid, even near-instantaneous, temperature rise of the liquid substrate. Compared to using tanks (generally 300 to 500 m³, with a residence time of 1 to 2 hours) equipped with steam injection nozzles, continuous flow mixing of the fluids allows for faster heating, reduced steam consumption, and reduced 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 continuous flow allows for an even greater reduction in the process's greenhouse gas emissions.

[0090] In a particularly preferred manner, gelatinization is carried out using a continuous direct steam injection device, preferably a jet-cooker (or cooker) device.

[0091] 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.

[0092] The gelatinized substrate can be used as a fermentation substrate, possibly after one or more additional treatments.

[0093] The liquid substrate, preferably gelatinized and preferably uncooled, comprising saccharidases, preferably α-amylases, may be subjected to a dextrinization step, for example, after being introduced into a tank in which the substrate is maintained for a certain period. The duration of the dextrinization is preferably 0.5 to 4 hours. This step allows the enzyme contained in the liquid substrate to continue hydrolyzing the starch. Advantageously, the dextrinization is carried out until a dextrose equivalent (DE) of 4 to 14 is obtained. The DE is an indicator of starch hydrolysis. At DE = 0, the starch is intact. At DE = 100, the starch is completely converted to glucose. The method used for measuring the DE is the Lane-Eynon method. A substrate is then obtained that is said to be "dextrinized".

[0094] The dextrinized substrate can be used as a fermentation substrate, possibly after one or more additional treatments.

[0095] 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 chosen from the group consisting of glucosidases. This step is called "saccharification" and allows the hydrolysis of dextrins into glucose. In the present In this text, the term "saccharification" is used to refer to any process of hydrolysis of dextrins into glucose, regardless of the degree of hydrolysis achieved; the saccharification stage may also be called "pre-saccharification" when the hydrolysis is not complete or nearly complete.

[0096] Prior to the introduction of the enzymes, the liquid substrate can be introduced into a tank.

[0097] The enzyme(s) introduced are preferably chosen from the group consisting of α,4-glucosidases, amylo-α,6-glucosidases, and mixtures thereof. More preferably, at least two enzymes are introduced into the liquid substrate; even more preferably, at least one α,4-glucosidase and one amylo-α,6-glucosidase are introduced into the liquid substrate. The α,4-glucosidase hydrolyzes the α-(1,4) bonds involved in the linear glucose chains of dextrins; the amylo-α,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, and preferably between 55 and 65°C. These conditions allow for optimal enzyme function. The quantity of enzymes introduced can range from 0.5 to 3.0 kg per tonne of assimilable carbohydrates.

[0098] Preferably, to carry out the saccharification, the liquid substrate comprising the glucosidases is kept in its container for a certain period of time. Preferably, the duration of the saccharification 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").

[0099] In some embodiments, dextrinization and saccharification may be simultaneous (at least in part).

[0100] A glucose-enriched substrate is obtained, usable as a fermentation substrate, either as is or after possible additional treatments, for example purification, in particular filtration and / or demineralization. "Glucose-enriched medium" means a medium in which the glucose concentration is higher than that of the substrate before saccharification.

[0101] Preferably, when the substrate undergoes a pre-saccharification step of 0.5 to 6h, it is not subjected to further purification.

[0102] The hydrolyzed substrate can be used as a fermentation substrate.

[0103] Advantageously, the preparation of the fermentation substrate as described above generates a greenhouse gas emission of less than 100 kg of CO2 oil equivalent (CO2eq) per tonne of plant material used, preferably less than 60 kg of CO2eq per tonne of plant material used, more preferably less than 40 kg of CO2eq per tonne of plant material used.

[0104] The hydrolyzed substrate advantageously comprises one or more of the following characteristics: • 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; • 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; • 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; • 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.

[0105] The amounts of protein, digestible carbohydrate, fat and fiber can be determined as indicated above.

[0106] According to the invention, the liquid substrate is contacted with at least one so-called "deviscosifying" enzyme, preferably at least two deviscosifying enzymes. "Deviscosifying enzymes" are understood to mean 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, and combinations thereof. Particularly preferred, the liquid substrate is contacted with at least one glucanase, preferably at least one beta-glucanase, and with at least one xylanase. The action of these enzymes is to hydrolyze the water-insoluble fibers to render them water-soluble. The amount of devicosating enzyme introduced into the liquid substrate can be from 0.5 to 4.0 kg per tonne of dry matter (of the liquid substrate).Preferably, at least one glucanase is introduced in an amount of 0.5 to 2.0 kg per tonne of dry matter and / or (preferably and) at least one xylanase in an amount of 0.5 to 2.0 kg per tonne of dry matter.

[0107] 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 after the formation of the liquid substrate up to the fermentation stage. Thus, the introduction of these enzymes can take place after a or several of the following steps: liquid substrate formation, hydrolysis, dextrinization, gelatinization, saccharification, introduction of amylases, introduction of glucosidases. Preferably, the deviscosifying enzymes are introduced before the gelatinization step, specifically after the formation of the liquid substrate (starch fluid) or after the introduction of saccharidases (preferably α-amylases).

[0108] The process according to the invention includes a step of fermenting the liquid substrate (preferably the hydrolyzed substrate). More particularly, this fermentation step comprises 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 tonne of dry matter of the liquid substrate.

[0109] In some embodiments, the microorganism is brought into contact with, as a fermentation substrate, a glucose-enriched substrate (i.e., one that has undergone the saccharification step) as described above. Preferably, the substrate is introduced into a tank and the microorganism is 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, preferably 4.0 to 4.5, during the fermentation step.

[0110] In other embodiments, the microorganism, preferably one or more yeasts, is brought into contact with, as a fermentation substrate, a dextrinized substrate as described above. 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,4-glucosidase and one amylo-α,6-glucosidase. This step is called the "propagation step." Advantageously, the substrate is introduced into a vessel, and the microorganism and the 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 partly carried out simultaneously.

[0111] In other embodiments, the microorganism, preferably one or more yeasts, is brought into contact with, as a fermentation substrate, a gelatinized substrate as described above. 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 group consisting of the glucosidases. Particularly preferred, the substrate is brought into contact with the microorganism and with at least one α-amylase, one α,4-glucosidase, and one amylo-α,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.

[0112] 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.

[0113] 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.

[0114] Preferably, the fermentation is carried out for a period of 36 to 70 hours, preferably 40 to 60 hours. Advantageously, at the end of the fermentation, all of 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)).

[0115] At the end of the fermentation, a fermented medium is obtained.

[0116] The process according to the invention includes a step of distilling 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 having a variable opening diameter connects the column and the condenser. The vapors passing beyond the diaphragm are then condensed by means of the condenser.

[0117] The distillation of a fermented medium to recover the alcohols produced during fermentation is 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 a distillation process is as follows. Upon boiling, the alcohol vapors reach the diaphragm, which is initially closed. The vapors are then condensed by means of 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 exchanges are 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.

[0118] 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).

[0119] 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°.

[0120] The process according to the invention allows obtaining a distillation residue containing a small amount of water-insoluble fibers.

[0121] The process according to the invention then comprises a solid-liquid separation step. Preferably, the solid-liquid separation is chosen from separations by centrifugation, separations by filtration, such as membrane filtration and / or Rotary drum filtration, settling, and combinations thereof are used in solid-liquid separation. This process can involve one or more of the aforementioned separation steps. Solid-liquid separation results in a solid fraction and a liquid fraction. Water-insoluble compounds are thus separated from the water-soluble compounds contained in the liquid fraction. Specifically, insoluble fibers are hydrolyzed, particularly during fermentation, using deviscosifying enzymes such as glucanases and xylanases, transforming them into soluble compounds that are transferred to the liquid phase and therefore removed during solid-liquid separation.

[0122] The solid fraction is collected. The solid fraction preferably comprises a dry matter content of at least 20% by weight, 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.

[0123] Advantageously, the protein isolate produced by the process according to the invention comprises: • a quantity of protein, relative to the total dry weight of the isolate, of 75 to 95% by weight, preferably 75 to 90% by weight, more preferably 75 to 85% by weight, even more preferably 78 to 85% by weight, for example 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 • an amount of digestible carbohydrate, relative to the total dry weight of the isolate, of 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; and / or • 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 • a quantity of fiber, relative to the total dry weight of the isolate, 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.

[0124] 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. Examples

[0125] The following examples illustrate the invention without limiting it.

[0126] In the examples, the following analysis and measurement methods were used: • Measurement of the quantity of protein: Kjeldahl method using a FOSS titrator according to ISO 5983-2; • Measurement of the quantity of lipids: AO AC 922.06 method; • Quantity of digestible carbohydrates: determined by calculating the difference between the total weight of the product and the sum of the weights of protein, fat, total fiber, water and ash; • Measurement of the quantity of ash: method according to the standard NF EN ISO 2171; • 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; • Measurement of the quantity of total fiber: according to the AO AC 2011.25 method; • Measurement of the alcohol content: using a densimeter.

[0127] Example 1: Production of a protein isolate from pea flour

[0128] 300 kg of peas are hulled using a PEDT 600 roller mill at STOLZ (cylinder spacing of 4.5 mm) and 30 kg of skins are separated from the almonds using an SNST 550 sieve from STOLZ equipped with a 5 mm sieve and a 2 mm sieve, and an SEP AIR 400 air separator from STOLZ (with an air flow rate of 2600 m3 / h).

[0129] 270 kg of almonds are ground in a ZPS 500 grinder from Alpine - Hosokawa used at a speed of 2650 rpm, in order to obtain micronized pea flour having a median volume diameter (D50) of particles less than or equal to 30 sqm. This flour is sent to an air separator using two successive selectors: a first Alpine - Hosokawa ZPS 500 selector operating at a rotation speed of 1600 rpm and with an air flow rate of 5300 m3 / h, and a second Alpine - Hosokawa ATP 315 selector operating at a rotation speed of 3400 rpm and with an air flow rate of 3600 m3 / h, in order to separate the protein-enriched fraction weighing 70 kg from the starch-enriched fraction weighing 200 kg. During aerodynamic separation, light particles with a D50 of 1 to 5 qm are carried to the top of the cyclone while heavier particles, with a D50 of 10 to 30 qm, are carried to the bottom.

[0130] 200 kg of the starch-enriched fraction are mixed with 600 litres of drinking water in an ISTILL 1000 reactor with a capacity of 1000 liters. The reactor is equipped of a distillation column, an electric resistance for heating the medium and a radiator supplied with cold water for cooling the medium.

[0131] The composition of the starch-enriched fraction is shown in Table 1 below (as mass percentage).

[0132] [Tables 1] Moisture 11.1% Protein 12.2% Fat 0.8% Digestible Carbohydrate 70.5% Fiber 3.6% Ash 1.8%

[0133] 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.

[0134] The mixture is heated to a temperature of 65°C using an electric heating element, and this temperature is maintained for 30 minutes. The mixture is then cooled to 32°C. An alpha-amylase (LPHERA® alpha-amylase, marketed by NOVOZYMES) is introduced into the medium at a concentration of 2.7 g / kg of digestible carbohydrates. At the same time, a mixture of glucosidases (Optidex® mixture, marketed by IFF, containing a mixture of an α-1,4-glucosidase and an amylo-α,6-glucosidase) is introduced into the medium at a concentration of 2.7 g / kg of digestible carbohydrates.

[0135] A Saccharomyces bayanus yeast is introduced at a concentration of 3.5 g / kg of dry matter of the mixture. 0.15 mL of vegetable oil-based antifoam is added per liter of reaction medium.

[0136] The medium is then cooled until it reaches a temperature of 25°C.

[0137] 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.

[0138] 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.

[0139] The composition of the product obtained is shown in Table 2 below, as a percentage on dry matter.

[0140] [Tables2] Protein 80.2% Digestible Carbohydrates 5.4% Fat 3.6% Total Fiber 8.9% Ash 1.9%

[0141] It is observed that a protein isolate comprising a very high protein content has been obtained.

[0142] Example 2: Production of a protein isolate according to other parameters

[0143] 130 kg of starch-enriched fraction obtained by dehulling, micronization and The aerodynamic separations 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-fed radiator for cooling the medium.

[0144] The starch-enriched fraction is as described in Example 1 and its composition is that shown in Table 1 above.

[0145] 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 dose of 3.9 g / kg of digestible carbohydrates. To solubilize (by hydrolysis) the fibers, beta-glucanase and xylanase are added at doses of 1.1 g / kg of dry matter of the mixture for beta-glucanase and 1.1 g / kg of dry matter of the mixture for xylanase.

[0146] The mixture is heated to a temperature of 65°C and this temperature is maintained for 30 minutes. The mixture is then cooled to 60°C. A mixture of glucosidases (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.

[0147] Following this residence time, the medium is cooled to a temperature of 30°C. A Saccharomyces cerevisiae yeast is introduced in a quantity of 1.4 g / kg of dry matter of the mixture. 20 mL of vegetable oil-based antifoam is added to the medium.

[0148] Fermentation is carried out by maintaining the medium at 30°C for 60 hours with pH adjustment using caustic soda to maintain it between 4.0 and 4.5. The alcohol content (in particular, 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.

[0149] After distillation, the distillation residue or "vinasse" has the following composition:

[0150] [Tables3] Protein 53.0% Digestible Carbohydrates 9.7% Fat 3.3% Total Fiber 31.8% Ash 2.2%

[0151] 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.

[0152] The composition of the product is shown in Table 3 below as a percentage on a dry matter basis.

[0153] [Tables4] Protein 80.8% Digestible Carbohydrates 5.1% Fat 2.9% Total Fiber 9.1% Ash 2.1%

[0154] The molecular weight distribution profile of the product proteins is 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 of the proteins 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.

[0155] [Tableaux5] >10 000 kD 33,0 % 5 000 - 10 000 kD 35,0 % 3 000 - 5 000 kD 13,0 % 1 000 - 3 000 kD 8,3 % 500 - 1 000 kD 1,9% < 500 kD 8,8 %

Claims

Demands

1. A process for preparing a protein isolate, comprising the following steps: • providing at least one seed containing starch and protein; • micronizing said at least one seed to obtain a micronized fraction; • treating the micronized fraction by air 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; • contacting the liquid substrate or the hydrolyzed substrate with at least one enzyme selected from the group consisting of xylanases, glucanases, and combinations thereof; • fermenting the hydrolyzed substrate to obtain a fermented medium;• Distillation of the fermented medium, so as to collect a distillate and a distillation residue; • Treatment of the distillation residue by solid-liquid separation, so as to obtain a liquid fraction and a solid fraction; • Collection of the solid fraction.

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 al,4-glucosidase and one amylo-al,6-glucosidase.

7. A method according to any one of claims 1 to 6, wherein the liquid substrate or the hydrolyzed substrate is contacted with at least one xylanase and at least one glucanase, preferably at least one beta-glucanase.

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 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 method 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 process according to any one of claims 1 to 11, comprising the production of a fermentation product during fermentation, said fermentation product being selected from alcohols, and preferably being ethanol.