Method for preparing a substrate for fermentation

Abstract

The invention relates to a process for preparing a fermentation substrate comprising the steps of: supplying at least one legume seed containing starch and protein; micronizing the seed to obtain a micronized fraction; purifying the micronized fraction to collect a starch-enriched, protein-depleted fraction; mixing the starch-enriched, protein-depleted fraction with a liquid to form a starch fluid; and hydrolyzing the starch by mixing steam with the starch fluid to obtain a hydrolyzed starch fluid. The invention also relates to a fermentation substrate, a fermentation process, and a fermentation product. Figure 1 illustrates this process.

Description

Title of the invention: Method for preparing a substrate for fermentation Scope of the invention

[0001] The present invention relates to methods for preparing a substrate usable in fermentation from a legume that emit low greenhouse gas emissions and to fermentation substrates obtained by such methods. Technical background

[0002] The fermentation industry, such as the production of ethanol, organic acids, amino acids, or vitamins, requires substrates rich in glucose or sucrose. Glucose is the basic food source for the microorganisms necessary for the production of the products mentioned above.

[0003] To obtain this glucose, the raw material used is primarily a carbohydrate-rich cereal such as wheat and corn or, when the substrate contains sucrose, sugar beet or sugar cane. When the raw material contains sucrose, it is conventionally extracted from the plant to obtain the sugary juice that will serve as a substrate for fermentation. When the raw material contains starch, the typical glucose production process includes a first step of producing a flour from this raw material, followed by a liquefaction (or hydrolysis) step to obtain a starch-rich milk, followed by saccharification to convert the starch into fermentable glucose that will serve as an ingredient in a fermentation substrate.

[0004] More specifically, when wheat is used as a source of glucose for an ethanol production process, the grain is first milled to separate the bran from the flour. The flour is then diluted with water to achieve approximately 30% dry matter, and an enzyme such as α-amylase is added to this mixture. The resulting milk is introduced into a tank containing steam injection nozzles. By injecting steam, the temperature of the milk is raised to approximately 95°C, causing the starch granules in the flour to burst and release the starch chains into the surrounding medium.Starch is a glucose polymer composed of a mixture of two homopolymers: amylose, a linear glucose polymer in which the glucose units are linked by α(1→4) bonds, and amylopectin, a branched glucose polymer in which the presence of α(1→6) bonds (in addition to α(1→4) bonds) results in numerous branches. The presence of α-amylase allows the hydrolysis of these chains into smaller fragments called dextrins. The size of the tank. The tank is generally sized to achieve a residence time of 1 to 2 hours, allowing sufficient contact time for the enzyme to break down the starch chains into dextrins. In one variation of the process, the resulting dextrin solution is cooled to 60°C before being transferred to another tank. There, another enzyme, such as glucoamylase, is added to hydrolyze the dextrins into glucose. This process, called pre-saccharification, typically takes between 3 and 4 hours, depending on the enzyme dosage, and provides the minimum glucose level necessary for microbial growth. The resulting glucose solution is then cooled again to approximately 30°C and transferred to a fermentation tank. In the case of ethanol production, yeast is added to this tank.These microorganisms will consume glucose and produce ethanol, intended in particular for the food processing, pharmaceutical and green chemistry industries.

[0005] According to a second embodiment of the process, pre-saccharification is not performed, but a propagation step is carried out. The dextrin solution obtained after liquefaction is directly cooled to 30°C before being injected into a tank. At the same time, the yeast and glucoamylase are injected. Since the temperature is 30°C, the glucoamylase has reduced activity, but it is still sufficient to provide the glucose necessary for yeast growth.

[0006] In cases where glucose from wheat is used in the production of organic acids, vitamins, or amino acids, the glucose production process is generally different. After the liquefaction step, the saccharification of the dextrins must be complete, unlike the process used for ethanol production. The saccharification time is between 40 and 60 hours to ensure that all the dextrins are converted into glucose. At the end of saccharification, the glucose content must be greater than 95% on a dry matter basis. For the production of these organic acids, vitamins, or amino acids, the glucose solution must be free of all insoluble species and contain the lowest possible level of soluble species other than glucose (such as proteins, fats, minerals, and other organic matter) to be used as a fermentation substrate.Purification steps are therefore usually carried out on the glucose solution, such as filtration, ion exchange and adsorption.

[0007] When the raw material is sugar beet, the sugar extraction process begins by washing the root to remove the rootlets. The washed beet is then cut into small strips called cossettes. These are introduced into a device, called a diffuser, containing a volume of water that circulates in a counter-current flow and is heated to 80°C. During this operation, the soluble compounds of the beet migrate into the water by a process of osmosis. The water, enriched with soluble compounds (the The "diffusion juice" exits at the top of the diffuser, and the sugar-depleted cossettes exit at the tail as pulp. The diffusion juices are then treated with lime (or liming) to precipitate some of the impurities present. Liming is generally followed by double carbonation (addition of CO2) to precipitate the remaining lime in the juice. The impurities and precipitated lime are then separated from the juice by filtration. The purified juice then undergoes an evaporation stage to concentrate it until a syrup with a sugar concentration close to saturation is obtained. Evaporation typically takes place in a multi-effect evaporator (several successive evaporators) in which the pressure is lowered from effect to effect to reduce the boiling point of the concentrated juice.The resulting syrup can then be used in a fermentation process, most often for the production of ethanol.

[0008] The processes described above are very high emitters of greenhouse gases, particularly carbon dioxide and nitrous oxide, due to the processes described above and the plants used. Indeed, these plants need to obtain significant quantities of mineral nitrogen from the soil to grow (2 to 3 kg per 100 kg of corn or wheat). This nitrogen is then supplied by nitrogen fertilizers, which constitute the main cause of greenhouse gas emissions related to the cultivation of these plants due to the significant emissions involved in the production of synthetic nitrogen (Haber-Bosch process) and the volatilization of nitrous oxide resulting from the application of the fertilizers.

[0009] There is therefore a real need to develop a process for preparing a substrate usable in fermentation, particularly for the industrial-scale production of ethanol or other organic compounds, with reduced greenhouse gas emissions. Summary of the invention

[0010] The invention relates primarily to a method for preparing a fermentation substrate, comprising the following steps: • the supply of at least one legume seed comprising starch and protein; • the micronization of said at least one seed, so as to obtain a micronized fraction; • the purification of the micronized fraction, so as to collect a fraction enriched in starch and depleted in protein; • mixing the starch-enriched, protein-depleted fraction with a liquid to form a starch fluid; and • the hydrolysis of starch by mixing steam with the starch fluid, so as to obtain a hydrolyzed starch fluid.

[0011] In embodiments, the process further comprises a step of introducing into the hydrolyzed starch fluid at least one enzyme selected from the group consisting of glucosidases, preferably at least two enzymes selected from the group consisting of glucosidases, more preferably at least one α-1,4-glucosidase and one amylo-α,6-glucosidase.

[0012] In embodiments, the process further includes a step of introducing into the starch fluid at least one enzyme chosen from the group consisting of saccharidases, preferably an α-amylase.

[0013] In embodiments, the legume seed includes a husk, and the process includes a step of dehulling the seed prior to micronization.

[0014] In some embodiments, the hydrolysis of starch is carried out using a continuous mode direct steam injection device.

[0015] In embodiments, the purification of the micronized fraction includes an aerodynamic separation step, preferably using a cyclone with selector.

[0016] In embodiments, the legume is chosen from the group of beans, peas, broad beans, lentils, chickpeas and mixtures thereof.

[0017] In embodiments, the process generates greenhouse gas emissions of less than 100, preferably 60, kg of CO2 oil equivalent per tonne of legume seed used.

[0018] The invention also relates to a fermentation substrate obtained by a process as described above.

[0019] The invention also relates to a fermentation process comprising bringing a fermentation substrate as described above into contact with at least one microorganism.

[0020] In embodiments, the microorganism is chosen from the group consisting of yeasts, bacteria and combinations thereof.

[0021] The invention also relates to a fermentation product obtained by a process as described above.

[0022] In embodiments, the fermentation product comprises at least one compound selected from the group consisting of alcohols, preferably ethanol, organic acids, amino acids, vitamins and mixtures thereof.

[0023] The present invention addresses the need expressed above. More particularly, it provides a process for preparing a fermentation substrate that is more environmentally friendly, and more specifically has low greenhouse gas emissions, while also enabling the production of a nutrient-rich substrate for fermentation, particularly one rich in glucose, and being capable of being produced at industrial scale.

[0024] This is achieved through the use of a specific raw material, namely a legume, and the combination of a micronization step of said legume with a purification step of the micronized legume, resulting in a fraction enriched in starch and depleted in protein. Brief description of the figures

[0025] [Fig-1] represents a schematic representation of an example device Direct steam injection in continuous mode usable in the invention. The arrows represent the direction of flow of the fluxes. Detailed description

[0026] The invention is now described in more detail and in a non-limiting manner in the following description.

[0027] Unless otherwise indicated, all percentages are mass percentages.

[0028] 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.

[0029] The invention relates to a method for preparing a substrate from at least one legume seed.

[0030] 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.

[0031] 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 and mixtures thereof.

[0032] 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 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 avoids 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.In addition, the nitrogen from the air fixed by the legumes is returned to the following crop via the decomposition of crop residues (aerial and underground parts) by Rhizobium bacteria, the most easily degradable residues (leaves, stems with little wood in carbon / nitrogen ratio). The lower part of the plant material (stems, roots) decomposes and releases nitrogen within a few weeks, while the woody parts (stems, roots) mineralize more slowly. Specifically, carbon emissions from legume cultivation are estimated at 200 kg of CO2 oil equivalent per tonne of legume. When legume cultivation is intercropped with cereal crops in a crop rotation system (for example, alternating peas, wheat, and oats), the application of nitrogen fertilizers is reduced, which can lower carbon emissions by 189 kg of CO2 oil equivalent per tonne of legume: in this case, legume cultivation therefore has a near-neutral net carbon balance of 11 kg of CO2 oil equivalent per tonne of legume.

[0033] Legume seeds are a plant material comprising a hull and a kernel. 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.

[0034] Preferably, a legume seed comprising a skin and an kernel is used as the starting raw material.

[0035] Advantageously, the process according to the invention includes a step of removing the hull (or hulling) from the legume seeds. The hull is composed mainly of insoluble fibers that are not consumed by fermentation microorganisms. Furthermore, since the majority of legume seed contaminants are found in the hull, its removal reduces the risk of contamination of the prepared substrate. In addition, the presence of fibers also increases the viscosity of the prepared fermentation substrate, which limits its dry matter content. The mass of fibers can, for example, represent 8 to 10% of the total dry matter of the legume seed.

[0036] Preferably the peeling is a mechanical peeling, carried out for example by abrasion, compression, impact, shearing or any other appropriate mechanical action.

[0037] Advantageously, dehulling is carried out by grinding the seed and then separating the resulting particles according to their size. Any suitable type of grinder can be used for grinding, in particular any grinder using one of the mechanical forces mentioned above. Specifically, a pendulum grinder using compression force can be used. After grinding, a mixture of hull fragments and almond powder (also referred to as flour in this text) is obtained.

[0038] The step of separating the obtained particles is preferably carried out by sieving. In particular, the particles can be separated by passing them through a sieve with a suitable mesh size, allowing the film fragments to be separated from the flour. By For example, particles smaller than a size between 200 and 600 µm can be separated from film fragments of a size greater than or equal to such a size.

[0039] The legume seed, preferably the hulled seed, more preferably the flour, is subjected to a grinding step and a ground fraction is collected.

[0040] Advantageously, the grinding process includes, or is, micronization, preferably by dry method. The resulting ground fraction is then a micronized fraction.

[0041] Legumes have a protein content on a dry matter basis that is significantly higher than that of plants traditionally used as raw materials in fermentation industries (cereals, beets, and sugarcane). For example, the protein content of legumes can reach up to approximately 30% by weight on a dry matter basis, compared to approximately 10 to 12% by weight for cereals, and considerably less for beets and sugarcane. Such a high protein content causes certain difficulties in conventional preparation processes. First, during the starch hydrolysis step, the applied temperature causes the proteins to coagulate, rendering them insoluble. A large quantity of insoluble proteins, combined with the presence of fiber, can lead to the formation of a magma that is difficult to transfer from one step to the next and creates a risk of blockage in the piping.Furthermore, the formation of a magma restricts the technologies that can be used during the hydrolysis step, complicating or preventing the use of certain devices, particularly continuous direct steam injection systems. Indeed, in such devices, the formation of a plug of proteins and fibers can lead to a significant increase in pressure within the device, causing damage (especially to the seals), or even an explosion, which could injure a nearby operator. In addition, a large quantity of protein also results in a high level of free amino acids. During fermentation, this high level of free amino acids can promote the growth of undesirable microorganisms, such as acetic acid bacteria instead of yeasts in the case of ethanol production.

[0042] The micronization step, in combination with the subsequent purification step, overcomes the aforementioned drawbacks caused by a large amount of protein. The micronization step detaches the proteins from the starch granules, allowing for their subsequent separation.

[0043] By "micronization" is meant a grinding process that produces particles with a median diameter by volume of less than 100 pm, preferably less than 50 pm, of preferably less than 30 pm. The median volume diameter (D50) of the particles can be measured according to standard NF ISO 13320-1.

[0044] 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.

[0045] Micronization is most preferably carried out at room temperature (i.e. between 15 and 30 °C).

[0046] Micronization has the additional advantage of being low emitter of greenhouse gases, preferably being carried out at ambient temperature and by dry method.

[0047] 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.

[0048] Following purification, a starch-enriched, protein-depleted fraction is collected. Preferably, a protein-enriched, starch-depleted fraction is also recovered. The term "starch-enriched, protein-depleted fraction" refers to a fraction in which the starch / protein molar ratio (on a dry matter basis) is greater than that of the purified fraction. The term "protein-enriched, starch-depleted fraction" refers to a fraction in which the starch / protein molar ratio (on a dry matter basis) is less than that of the purified fraction.

[0049] 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 can advantageously be used. Preferably, the separation is an air separation. "Air separation" means 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 a cyclonic separation. It can be carried out using a cyclone, advantageously combined with a selector. "Selector" means any variable-speed rotating element equipped with radial blades 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, at one end of the device (protein-enriched and starch-depleted fraction), while heavier particles are collected at the other end (starch-enriched and protein-depleted fraction).

[0050] Preferably, the starch-enriched and protein-depleted fraction comprises a The quantity of carbohydrates greater than or equal to 40% by weight, preferably 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 quantity of carbohydrates in the recovered starch-enriched, protein-depleted fraction may include 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.

[0051] Preferably, the starch-enriched, protein-depleted 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, protein-depleted 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.

[0052] Preferably, the starch-enriched, protein-depleted 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 weight of the fraction; in particular, the fraction may comprise a fiber content 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. "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.

[0053] Preferably, the collected starch fraction 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.

[0054] According to the process of the invention, the starch-enriched and protein-depleted fraction is then mixed with a liquid to form a starch fluid (i.e., a fluid containing starch). The liquid is preferably water. The starch fluid is preferably in the form of a dispersion, and even more preferably it is of a starch milk. By "starch milk", we mean a suspension of starch in water (said suspension may include other components, solubilized in water or not).

[0055] Preferably, 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.

[0056] Advantageously, at least one enzyme is introduced into the starch fluid (preferably milk starch). The enzyme is preferably a saccharidase, and more particularly an α-amylase. Most preferably, the enzyme is thermostable, particularly at temperatures of 90 to 130°C. The amount of enzyme added is preferably 0.2 to 0.8 kg of enzyme per tonne of dry starch, and more preferably 0.3 to 0.5 kg of enzyme per tonne of dry starch. The pH of the starch fluid is preferably adjusted to a pH between 3.5 and 6.5, and 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. However, at this stage of the process, α-amylases do not have access to the starch molecules which are enclosed in granules.

[0057] The starch fluid (preferably starch milk) is then subjected to a starch hydrolysis step. The purpose of this step is to cause the starch granules to burst in order to release the starch molecules into the fluid, thus enabling the action of enzymes (this step, which aims to cause the starch granules to burst, can also be called "liquefaction"). The bursting of the starch granules is achieved by heating the starch fluid (preferably starch milk) to a temperature that allows the liquid to be introduced into the granule, causing the granule to swell and then burst. This yields a hydrolyzed starch fluid.

[0058] According to the invention, hydrolysis is carried out by mixing water vapor with the starch fluid, preferably using a direct steam injection device.

[0059] Preferably, the starch fluid is heated (by mixing with steam) to a temperature (referred to in this text as hydrolysis temperature or liquefaction temperature) of 90 to 130 °C, preferably 90 to 100 °C.

[0060] Particularly advantageously, the hydrolysis is carried out by mixing a stream of water vapor with a stream of the starch fluid. By "stream" is meant a fluid (gas or liquid) in motion.

[0061] 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 both fluids, into the mixer is carried out at least partly simultaneously with the discharge of the mixer of said mixture.

[0062] Mixing steam with the starch fluid in a continuous flow allows for a very rapid, even near-instantaneous, temperature increase of the starch fluid. Compared to the use of tanks (generally 300 to 500 m³, with a residence time of 1 to 2 hours) equipped with steam injection lances, which are conventionally used, 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 hydrolysis step by continuously mixing steam with the starch fluid in a continuous flow allows for an even greater reduction in the process's greenhouse gas emissions.

[0063] Most preferably, the hydrolysis is carried out using a continuous direct steam injection device, more preferably a jet-cooker (or cooker) device. An example of such a device is shown in [Fig. 1].

[0064] Preferably, the continuous-mode direct steam injection device comprises a starch fluid supply line 1 for introducing the fluid into a tube 3 called the "mixing tube," and a steam supply line 2 terminated by a steam injector 4 for injecting the steam into the mixing tube 3. Preferably, the steam and starch fluid injection points in the mixing tube 3 are arranged coaxially. Advantageously, the steam injector 4 forms a needle in the starch fluid supply line 1. The starch fluid and steam flows are mixed within the mixing tube 3. The mixing tube 3 includes a fluid outlet 5, connected to a discharge pipe for releasing the mixture. The starch fluid and steam flows can be adjusted independently of each other, for example, by means of valves.

[0065] Preferably, the pressure of the starch fluid injected into the mixing tube is 0.2 to 1 MPa, more preferably 0.4 to 0.7 MPa. In particular, the pressure of the injected starch fluid can be 0.2 to 0.4 MPa, or 0.4 to 0.5 MPa, or 0.5 to 0.6 MPa, or 0.6 to 0.7 MPa, or 0.7 to 0.8 MPa, or 0.8 to 1 MPa. The pressure of the steam injected into the mixing tube is advantageously 0.4 to 1.5 MPa, more preferably 0.6 to 1 MPa. Specifically, the pressure of the injected steam can be 0.4 to 0.6 MPa, or 0.6 to 0.8 MPa, or 0.8 to 1 MPa, or 1 to 1.2 MPa, or 1.2 to 1.5 MPa. The pressure of the mixture at the outlet of the mixing tube is preferably 0.1 to 0.5 MPa, and even more preferably 0.2 to 0.4 MPa. In particular, The pressure of the mixture at the outlet of the mixing tube can be 0.1 to 0.2 MPa, or 0.2 to 0.3 MPa, or 0.3 to 0.4 MPa, or 0.4 to 0.5 MPa. Preferably, the pressure difference between the pressure of the mixture at the outlet of the mixing tube and the pressure of the starch fluid injected into the mixing tube is 0.1 to 0.5 MPa, preferably 0.2 to 0.4 MPa. This pressure difference can, for example, be 0.1 to 0.2 MPa, or 0.2 to 0.3 MPa, or 0.3 to 0.4 MPa, or 0.4 to 0.5 MPa. Such pressure ranges allow for optimal homogenization of the steam / starch fluid mixture.

[0066] Preferably, the outlet pipe has the following geometry: • a nominal diameter DN over a pipe length of 50 to 200 mm after the fluid outlet, then • a diameter DI equal to 1.5 to 2.5 times the diameter DN over a pipe length equal to 10 to 30 times the diameter DN, then • a diameter D2 equal to 2 to 5 times the diameter DN.

[0067] Such a geometry makes it possible to control the rise in viscosity caused by the swelling of the granules which takes place before they burst and thus to limit the risks of deterioration and explosion of the device.

[0068] The hydrolyzed starch fluid, preferably uncooled, can then be introduced into a tank. The residence time of the fluid in the tank is preferably 1 to 4 hours. This step, called dextrinization, allows the enzyme contained in the fluid to continue hydrolyzing the starch. Advantageously, dextrinization is carried out until a dextrose equivalent (DE) of 12 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.

[0069] The hydrolyzed starch fluid can be used as a fermentation substrate, possibly after one or more additional treatments.

[0070] The process according to the invention may in particular include a step of introducing at least one enzyme into the hydrolyzed starch fluid. This step is called "saccharification" and enables the hydrolysis of dextrins into glucose. In the present 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.

[0071] Preferably, prior to the introduction of the enzymes, the hydrolyzed starch fluid is introduced into a tank.

[0072] The enzyme or enzymes introduced are preferably chosen from the group consisting of glucosidases. More preferably, at least 2 enzymes are introduced into The hydrolyzed starch liquid is preferably further enriched with at least one α,4-glucosidase and one amylo-α,6-glucosidase. The α,4-glucosidase hydrolyzes the α-(α,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, preferably between 55 and 65°C. These conditions allow for optimal enzyme function. The quantity of enzymes introduced can be from 0.2 to 0.6 kg per tonne of dry starch.

[0073] In some embodiments, the duration of the saccharification is from 2 to 6 hours, preferably from 3 to 4 hours (in these embodiments, this step is more particularly referred to as "pre-saccharification").

[0074] In other embodiments, the saccharification time is from 30 to 70 hours, preferably from 40 to 60 hours. Advantageously, the amount of glucose in the medium after such a saccharification step is from 90 to 99% by weight, preferably from 92 to 97% by weight. The amount of glucose is determined according to standard NF EN ISO 10504.

[0075] A glucose-enriched medium is obtained, usable as a fermentation substrate, either as is or after possible additional treatments, for example purification, in particular filtration and / or demineralization. A "glucose-enriched medium" is understood to mean a medium in which the glucose concentration is higher than that of the hydrolyzed fluid before saccharification.

[0076] Preferably, when the substrate undergoes a pre-saccharification step of 2 to 6 hours, it is not subjected to further purification.

[0077] Preferably, when the substrate undergoes a saccharification step of 30 to 70h, it undergoes at least one subsequent purification step, preferably filtration and demineralization, preferably using ion exchange resins.

[0078] Advantageously, the process for preparing a fermentation substrate according to the invention generates a greenhouse gas emission of less than 100 kg of CO2 oil equivalent (CO2eq) per tonne of legume seed used, preferably less than 60 kg of CO2eq per tonne of legume seed used, more preferably less than 40 kg of CO2eq per tonne of legume seed used (for example, the process for preparing a fermentation substrate according to the invention can generate a greenhouse gas emission of 0 to 20, or 20 to 30, or 30 to 40, or 40 to 50, or 50 to 60, or 60 to 80, or 80 to 100, kg of CO2eq per tonne of legume seed used). Greenhouse gas emissions can be determined as shown in the Examples section below.

[0079]

[0080]

[0081]

[0082]

[0083] The invention also relates to a fermentation substrate obtained by, or capable of being obtained by, a preparation process as described above. The fermentation substrate according to the invention advantageously comprises one or more of the following characteristics (in particular when obtained by a process including a pre-saccharification step): • 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; • a quantity of 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. The amounts of protein, carbohydrate, fat and fiber can be determined as indicated above. Alternatively, the fermentation substrate according to the invention may advantageously comprise one or more of the following characteristics (in particular when obtained by a process including a saccharification step of 30 to 70 hours): • a quantity of carbohydrate, relative to the total dry weight of the substrate, greater than or equal to 90% by weight, preferably greater than or equal to 95% by weight; • a quantity of protein, relative to the total dry weight of the substrate, less than or equal to 500 ppm by weight, preferably less than or equal to 200 ppm by weight, more preferably less than or equal to 100 ppm by weight; • an amount of ash (i.e. all the minerals, including NaCl and CaCl2), relative to the total dry weight of the substrate, less than or equal to 200 ppm by weight, preferably less than or equal to 100 ppm by weight, more preferably less than or equal to 50 ppm by weight. The invention also relates to a fermentation process comprising the introduction contact of a fermentation substrate as described above with at least one microorganism. The microorganism is preferably chosen from the group consisting of yeasts, bacteria and combinations thereof.

[0084] In some embodiments, the microorganism is brought into contact with, as a substrate, a glucose-enriched medium (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 30°C, prior to its contact with the microorganism.

[0085] In other embodiments, the microorganism, preferably one or more yeasts, is brought into contact with, as a substrate, a hydrolyzed starch fluid (i.e., one that has not undergone the saccharification step) as described above. Preferably, the hydrolyzed starch fluid is pre-cooled to a temperature of 20 to 40°C, more preferably 25 to 35°C, and more preferably 26 to 30°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 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 saccharification step.

[0086] The invention also relates to a fermentation product obtained by, or capable of being obtained by, a fermentation process as described above. Preferably, the fermentation product comprises (or consists of) at least one compound selected from the group consisting of alcohols, preferably ethanol, organic acids, amino acids, vitamins, and mixtures thereof.

[0087] Preferably, when the fermentation product is or comprises a compound selected from organic acids, amino acids, vitamins and mixtures thereof, the fermentation process uses a substrate that has been prepared by a process comprising a saccharification step of 30 to 70 hours.

[0088] Preferably, when the fermentation product is or comprises a compound selected from alcohols, and is in particular ethanol, the fermentation process uses a substrate prepared by a process comprising a pre-saccharification step, or a hydrolyzed starch fluid is used as the substrate (in the latter case, the fermentation process very preferably comprises a step propagation as described above). Examples

[0089] The following examples illustrate the invention without limiting it.

[0090] A process for producing ethanol by fermentation using a substrate prepared from a legume (pea) according to the invention has been compared to a process for producing ethanol by fermentation using a substrate prepared from a cereal (wheat) and to a process for producing ethanol by fermentation using a substrate prepared from a beet.

[0091] Process for producing ethanol from beetroot

[0092] Once harvested, the beets are quickly transported and processed. The beets are then washed with water to remove all traces of soil, grass or rocks from the harvest.

[0093] Once removed from the washing plant, the beets are cut into cossettes (whose shape resembles French fries). The cossettes are 5 to 6 cm long and 2 to 3 mm wide. This cutting is done using a root cutter of the Model 2000 - 600 - 60 type from the company MAGUIN.

[0094] The beet slices are then conveyed to the diffuser, where they circulate counter-currently with hot water at 80°C. In this water, the soluble compounds of the beet migrate to produce a diffusion juice that exits at the top of the diffuser. The beet slices exit the diffuser as pulp, and some of the water they contain is removed by pressing or dehydration for recycling. The pulp can then be used in animal feed.

[0095] The diffusion juice then undergoes purification by treatment with quicklime, resulting in the precipitation of some of the impurities. The quicklime is obtained by calcining limestone at 900°C at a rate of 4.6 kg of limestone per tonne of beet. The CO2 produced during this operation is subsequently used in the diffusion juice to precipitate the slaked lime (Ca(OH)2) into calcium carbonate. The impurities and the precipitated lime are separated from the juice by filtration using a filter press.

[0096] The purified juice contains 85% water by weight. It is evaporated to concentrate it, resulting in a syrup with a sucrose concentration close to saturation, i.e., 60 to 70% by weight. Evaporation takes place in a multi-effect evaporator, with the pressure being reduced from effect to effect to lower the boiling point of the concentrated juice, thus preventing it from cooking. Concentrating the juice prevents sugar fermentation and allows the juice to be stored before being transported to the distillery.

[0097] The fermentation stage begins with the preparation of the pre-fermentation syrup The sugar (sucrose) is diluted to 7% by weight by adding water and then introduced into a tank called a pre-fermenter containing a solution of Saccharomyces cerevisiae yeast. The quantity of yeast is 6 g per ton of sugar. Urea, at a rate of 1.3 kg per ton of sugar, is added, as well as nitric acid to maintain the pH between 5.0 and 5.5. The temperature is adjusted between 30 and 35°C. The residence time is set between 4 and 5 hours, and a continuous flow of air is injected into the medium by means of a diffuser installed at the bottom of the tank.

[0098] When the alcohol content (in particular, ethanol) reaches a value between 6 and 8% by weight (as measured using a hydrometer), the so-called weak must is introduced into a fermentation tank. A sugar solution with a concentration between 20 and 25% (strong must) is added in a proportion of 30 to 50% by weight of the weak must. The exothermic alcoholic fermentation continues while the temperature is maintained between 30 and 35°C by means of a plate heat exchanger connected to a cooling tower. The fermentation takes place over a period of 30 to 40 hours. During this fermentation, the pH tends to decrease. A sodium hydroxide solution is therefore added over time to maintain the pH between 5.0 and 5.5. When all the sugar is converted into ethanol and carbon dioxide, the alcohol level in the fermenter is between 10 and 14% by weight.

[0099] The fermentation must is then subjected to a distillation step consisting of separating the alcohol fraction from the fermentation must. The latter is introduced into the middle of a vacuum distillation column containing several trays and then falls to the bottom of the column. The fluid is then heated to the boiling point of the water and ethanol mixture, at a temperature between 82 and 85°C, by means of a heat exchanger supplied with steam from a boiler. The alcohol vapors are collected at the top of the column with an alcohol content between 93 and 95% by weight.

[0100] The distillate obtained contains many impurities such as volatile compounds and other types of alcohol such as methanol, and is therefore purified by passing through the following different columns, which taken together form the rectification step: • Extraction column: In the column, water is added to the distillate from the distillation column. The difference in volatility of the compounds present in the distillate allows them to be separated. The highly volatile compounds, which are poorly soluble in water, are carried to the top of the column, while the water-soluble ethanol and methanol are carried to the bottom. • Rectification column: The alcohol-water mixture from the extraction column is distilled again until it reaches almost the azeotropic alcohol / water mixture (97% alcohol by weight) at the bottom of the column, while the impurities are collected at the top of the column (distillate). The impurities... The contents of the previous column are introduced into this distillate. • Demethylation column: This large column, containing a very large number of trays, separates methanol and other impurities from the ethanol. The resulting product is called "superfine alcohol." The residue containing methanol is mixed with the impurities collected from the previous step. • Head column: All impurities from the previous columns are passed through this column to purify them and produce fusel oils. The ethanol recovered at the bottom of the column is recycled to the extraction or rectification column to improve the ethanol purification yield. The fusel oils are collected at the top of the column.

[0101] All the rectification operations use the liquid separation process by vaporization-condensation fractionation.

[0102] During the distillation step, an insoluble residue is collected along with the unevaporated water. This residue is called vinasse. The dry matter content is approximately 6% by weight. As this dry matter content is too low, the residue is introduced into a falling-flow tubular evaporator. Evaporation takes place under vacuum (0.02 MPa pressure) with steam injection. The final dry matter content of the residue is 30 to 35% by weight. The concentrated vinasse can be used in agriculture, particularly for the preparation of a soil amendment.

[0103] For one unit of ethanol production, the quantity of CO2 equivalent in oil (CO2 eq) was estimated. The quantity of CO2 equivalent in oil represents all greenhouse gas emissions (including nitrous oxide, methane, etc., in addition to CO2). Its calculation is carried out using "emission factors" whose values ​​are obtained from the following databases: • EUROPEAN COMMISSION: Note on the conducting and verifying actual calculations of the GHG emission savings, 2015; • INSTITUT FÜR ENERGIE- UND UMWELTFORSCHUNG HEIDELBERG (IFEU): Biograce. Harmonized calculations of biofuel greenhouse gas emissions in Europe. - www.biograce.net; • BUNDESANSTALT FÜR LANDWIRTSCHAFT UND ERNÀHRUNG (BLE): Leitfaden Nachhaltige Biomassehstellung, 1st Edition, Bonn, 2010; • 1INAS INTERNATIONALES INSTITUT FÜR NACHHALTIGKEIT-SANALYSEN UND -STRATEGIEN, ÔKO-INSTITUT EV INSTITUT FÜR ANGEWANDTE ÔKOLOGIE EV, Gémis, 2014.

[0104] The results for the process of producing ethanol from a beet-derived substrate described above are presented in the table below. Ethanol production is 88,830 t / year (from 1,189,256 t of beet). The CO2eq emitted during Sugar extraction is attributed at a rate of 94% by weight to the production and processing of sugar juice and at a rate of 6% by weight to the production of pulp (which is indicated under the heading "considered yield" in the table below).

[0105] [Tables 1] Quantity (A) Emission factor (kg CO2eq / unit) (B) kg CO2eq (AxB) Agricultural emissions Seed (kg / Ha / year) 6 3.54 366828 Nitrogen fertilizer (kg / Ha / year) 119.7 5.88 12155695 P2O5 fertilizer (kg / Ha / year) 59.7 1.01 1041268 K2O fertilizer (kg / Ha / year) 134.9 0.58 1351289 CaO fertilizer (kg / Ha / year) 400 0.13 898073 Pesticides (kg / Ha / year) 1.3 10.97 16703745 Nitrous oxide (kg CO2eq / kg N) 119.7 8.08 16703745 Diesel (agriculture) (L / Ha / year) 175.9 3.14 9539021 Diesel (transport) (L / Ha / year) 67 3.14 10424820 Subtotal (kg CO2eq / t ethanol) Efficiency considered: 94% 558 Sugar extraction emissions Natural gas (Mj / year) 442377866 0.067 29639317 Electricity (kW / year) produced by coal-fired power plant 17856000 0.61 10892160 Limestone (kg / year) 54985000 0.00972 534454 Water (kg / year) 507018000 0.0004 202807 Water treatment (kg / year) 763269000 0.00027 206083 Subtotal (kg CO2eq / t ethanol) 439 Ethanol production emissions Natural gas (Mj / year) 731256874 0.067 48994211 Electricity (kW / year) 30153046 0.61 18393358 Nitric acid at 65% by weight (kg / year) 144324 1.89 Sodium hydroxide at 50% by weight (kg / year) 731600 0.47 343852 Yeast (kg / year) 1193 3.2 3818 Urea (kg / year) 366268 0.81 296677 Water (kg / year) 137514574 0.0004 55006 Water treatment (kg / year) 212241923 0.00027 57305 Subtotal (kg CO2eq / t ethanol) 770 Total process Total (kg CO2eq / t ethanol) 1767 Total (kg CO2eq / t ethanol) excluding vinasse 990

[0106] The total quantity of CO2eq calculated for the entire process is allocated as follows: 56% by weight of the total is allocated to ethanol production and 44% by weight of the total is allocated to the production of the co-product vinasse. The share of greenhouse gas emissions corresponding to ethanol production alone is indicated in the table above in the row "Total (kg CO2eq / t ethanol) excluding vinasse".

[0107] Process for producing ethanol from wheat.

[0108] The wheat grains are harvested. Unlike sugar beets, harvested wheat can be stored and kept for several months. Upon arrival at the industrial site, the wheat is first cleaned to remove stones and plant residues from the harvest.

[0109] The cleaned wheat is then ground using a roller mill, and then sifted to separate the bran (wheat hull) from the flour (kernel powder). These steps up to the collection of the flour are carried out dry and at ambient temperature.

[0110] The flour is then suspended in water to obtain a dry matter content of between 25 and 35% by mass. The resulting fluid is introduced into an 80 m³ tank equipped with steam injection lances. Simultaneously, an alpha-amylase enzyme (maxamyl HT) is added in a quantity of between 0.2 and 0.6 kg per tonne of flour. The pH is adjusted to between 5.0 and 6.5 by adding sulfuric acid. Steam is injected into the fluid using the injection lances until the fluid reaches a temperature between 90 and 95°C. This temperature allows water to penetrate the starch granules until the protective membrane of the granules ruptures. The starch released into the fluid is then hydrolyzed by the enzyme. The residence time is between 4 and 5 hours so that the starch is sufficiently broken down into dextrin chains.

[0111] The hydrolyzed must is sent to a 50 m3 stirred tank and the enzyme Deltazyme GA LE-5, containing an alpha 1-4 glucosidase and an alpha 1-6 glucosidase, is added to carry out saccharification. The residence time in the tank is 2 hours.

[0112] The following steps (fermentation, distillation and rectification) are identical to those described above for the process of producing ethanol from beet.

[0113] The quantity of oil equivalent CO2 emitted by the process was estimated as indicated above and the results are presented in the table below. Ethanol production is 23,887 t / year (from 80,235 t of wheat). The CO2eq emitted up to (not including) the saccharification stage is attributed 84% to the production and processing of flour and 16% to the production of fiber (bran) (which is indicated under the heading "considered yield" in the table below).

[0114] [Tables2] Quantity (A) Emission factor (kg CO2eq / unit) (B) kg CO2eq (AxB) Agricultural emissions Wheat crop (t / year) 80235 300 24070500 Diesel (transport) (L / Ha / year) 15 3.14 539867 Subtotal (kg CO2eq / t ethanol) Yield considered: 84% 865 Emissions from fermentation substrate production Electricity (kW / year) produced by nuclear power plant 4477249 0.04 179090 Subtotal (kg CO2eq / t ethanol) 439 Emissions from ethanol production Natural gas (Mj / year) 351254862 0.067 23534076 Electricity (kW / year) 12241584 0.04 489663 Sulfuric acid 96% by weight (kg / year) 1730122 0.21 363326 Sodium hydroxide at 50% by weight (kg / year) 157204 0.47 73886 Yeast (kg / year) 856 3.2 2739 Ammonia at 24% by weight (kg / year) 12755 2.7 234439 Water (kg / year) 226800000 0.0004 90720 Water treatment (kg / year) 239400000 0.00027 64638 Subtotal (kg CO2eq / t ethanol) 1032 Total process Total (kg CO2eq / t ethanol) 1904 Total (kg CO2eq / t ethanol) excluding vinasse 1066

[0115] The total quantity of CO2eq calculated for the entire process is allocated as follows: 56% by weight of the total is allocated to ethanol production and 44% by weight of the total is allocated to the production of the co-product vinasse. The share of greenhouse gas emissions corresponding to ethanol production alone is indicated in the table above in the row "Total (kg CO2eq / t ethanol) excluding vinasse".

[0116] Process for producing ethanol from peas.

[0117] Once harvested, the peas are dehulled by crushing using a pendulum crusher employing compression force, then the particles produced are passed through a sieve to separate the husks smaller than 500 pm from the husks larger than or equal to 500 pm.

[0118] The selected particles (less than 500 pm) are then subjected to a micronization step using an impact mill (so as to achieve a median volume diameter (D50) of the particles less than or equal to 30 pm). At the mill outlet, the resulting powder is introduced into turbo-cyclones with a selector, fed by an airflow to activate a cyclonic effect, in order to separate the particles according to their density. The lighter particles (called the protein fraction), having a D50 of 1 to 5 pm, are carried to the top of the cyclone, while the heavier particles, having a D50 of 10 to 30 pm, are carried to the bottom.

[0119] The heavy fraction (called the starch fraction) is recovered and mixed with water to obtain a fluid having a dry matter mass content of approximately 30%. The pH is adjusted between 4.0 and 6.0 by sulfuric acid, and then an alpha amylase type enzyme (maxamyl HT) is added to the fluid in an amount of 0.3 to 0.5 kg per tonne of dry starch matter.

[0120] In order to cause the starch granules it contains to burst, the fluid is then heated to 100°C by direct, continuous steam injection into a Hydro-thermal K510 type jet cooker. (fluid pressure including starch granules: 0.6 MPa; water vapor pressure: 0.9 MPa; fluid / vapor mixture pressure at jet-cooker outlet: 0.25 MPa).

[0121] The fluid is then subjected to a dextrinization step: for this, the uncooled fluid is placed in a 30 m³ tank for 4 hours. The fluid, cooled to 60°C, is then introduced into another tank to undergo a pre-saccharification step. An α,4-glucosidase and an amylo-α,6-glucosidase (Deltazyme GA LE-5) are introduced into the fluid at a rate of 0.3 kg per tonne of dry starch, and the pH is adjusted to a value between 4.0 and 4.5 with sulfuric acid. The fluid is left in the tank for 3 hours.

[0122] The subsequent steps (fermentation, distillation, and rectification) are identical to those described above for the process of producing ethanol from beet, except that the distillation column is not heated by a heat exchanger supplied with steam from a boiler, but by the vapors from the evaporator used to concentrate the vinasse, recompressed using a mechanical recompressor. The energy used in this case is electricity, not gas.

[0123] The quantity of oil equivalent CO2 emitted by the process was estimated as indicated above and the results are presented in the table below. Ethanol production is 7465 t / year (from 32982 t of wheat). The CO2eq emitted up to (not including) the pre-saccharification stage is attributed as follows: 67.2% by weight to the production and processing of the starch fraction, 24% by weight to the production of the protein fraction, and 8.8% to the production of fiber (husk) (which is indicated under the heading "considered yield" in the table below).

[0124] [Tables3] Quantity (A) Emission factor (kg CO2eq / unit) (B) kg CO2eq (AxB) Agricultural emissions Pea cultivation (t / year) 32982 200 6596400 Nitrogen fertilizer avoided 32982 -189 -6233598 Diesel (transport) (L / Ha / year) 6.3 3.14 163112 Subtotal (kg CO2eq / t ethanol) Considered yield of 67.2% 70 Emissions from fermentation substrate production Electricity (kW / year) produced by nuclear power plant 6523100 0.04 260924 Subtotal (kg CO2eq / t ethanol) 35 Emissions from ethanol production Natural gas (MJ / year) 78659000 0.067 5270153 Electricity (kW / year) 4116000 0.04 164640 Sulfuric acid at 96% by weight (kg / year) 39900 0.21 8379 Sodium hydroxide at 50% by weight (kg / year) 47161 0.47 22166 Yeast (kg / year) 256 3.2 819 Ammonia at 24% by weight (kg / year) 38068 2.7 102784 Water (kg / year) 67200000 0.0004 26880 Water treatment (kg / year) 84000000 0.00027 22680 Subtotal (kg CO2eq / t ethanol) 753 Total process Total (kg CO2eq / t ethanol) 858 Total (kg CO2eq / t ethanol) excluding vinasse 481

[0125] The total quantity of CO2eq calculated for the entire process is allocated as follows: 56% by weight of the total is allocated to ethanol production and 44% by weight of the total is allocated to the production of the co-product vinasse. The share of greenhouse gas emissions corresponding to ethanol production alone is indicated in the table above in the row "Total (kg CO2eq / t ethanol) excluding vinasse".

[0126] The results are summarized in the table below (only the shares of emissions attributed to ethanol production are considered):

[0127] [Tables4] kg of CO2 equivalent emitted per tonne of ethanol produced Wheat process (comparative) Beet process (comparative) Pea process (invention) Process up to distillation (not included) 488 559 60 Process from distillation 578 431 421 Total 1066 990 481

[0128] It is observed that the ethanol production process according to the invention emits much less greenhouse gas than comparative processes using a substrate prepared from cereal and beet.

[0129] Moreover, with regard to the process up to distillation, the process according to the invention produces very low greenhouse gas emissions.

Claims

Demands

1. A process for preparing a fermentation substrate, comprising the following steps: • supplying at least one legume seed comprising starch and protein; • micronizing said at least one seed, so as to obtain a micronized fraction; • purifying the micronized fraction, so as to collect a starch-enriched and protein-depleted fraction; • mixing the starch-enriched and protein-depleted fraction with a liquid, so as to form a starch fluid; and • hydrolyzing the starch by mixing steam with the starch fluid, so as to obtain a hydrolyzed starch fluid.

2. A process according to claim 1, further comprising a step of introducing into the hydrolyzed starch fluid at least one enzyme selected from the group consisting of glucosidases, preferably at least two enzymes selected from the group consisting of glucosidases, more preferably at least one al,4-glucosidase and one amylo-al,6-glucosidase.

3. A method according to claim 1 or 2, further comprising a step of introducing into the starch fluid at least one enzyme selected from the group consisting of saccharidases, preferably an α-amylase.

4. A process according to any one of claims 1 to 3, wherein the legume seed comprises a husk, and wherein the process comprises a step of dehulling the seed prior to micronization.

5. A method according to any one of claims 1 to 4, wherein the purification of the micronized fraction includes an aerodynamic separation step, preferably by means of a cyclone with selector.

6. A method according to any one of claims 1 to 5, wherein the hydrolysis of starch is carried out using a continuous mode direct steam injection device.

7. A method according to any one of claims 1 to 6, wherein the legume is chosen from the group of beans, peas, broad beans, lentils, chickpeas and mixtures thereof.