Method for preparing a substrate for fermentation

Abstract

The invention relates to a method for preparing a fermentation substrate, said method comprising the steps of: providing at least one leguminous plant seed comprising starch and proteins; micronizing the 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 hydrolysing the starch by mixing water vapour with the starch fluid so as to obtain a hydrolysed starch fluid. The invention also relates to a fermentation substrate, to a fermentation method, and to a fermentation product.

Description

[0001]Method for preparing a substrate for fermentation Field of the invention The present invention relates to methods for preparing a substrate usable in fermentation from a legume which emit low greenhouse gas emissions and fermentation substrates obtained by such methods. Technical background 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 of the microorganisms necessary for the production of the products mentioned above. To obtain this glucose, the raw material used is mainly a cereal rich in carbohydrates such as wheat and corn or, when the substrate contains sucrose, beet or sugar cane.When the raw material contains sucrose, it is conventionally extracted from the plant to obtain the sweet juice that will serve as a substrate for fermentation. When the raw material contains starch, the typical glucose production process involves 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. Specifically, when wheat is used as a glucose source for an ethanol production process, the grain is first ground to separate the bran from the flour. The flour is then diluted in water to reach about 30% dry matter, and an enzyme such as α-amylase is added to this mixture.The milk thus obtained is introduced into a tank containing steam injection pipes. By injecting steam, the temperature of the milk will increase to approximately 95°C, so that the starch granules contained in the flour burst and release the starch chains into the environment. Starch is a glucose polymer composed of a mixture of two homopolymers: amylose, which is a glucose polymer in linear form in which the glucose units are linked by α(1→4) bonds, and amylopectin, which is a glucose polymer in a so-called "branched" or ramified form, in which the presence of α(1→6) bonds (in addition to α(1→4) bonds) generates a large number of branches. The presence of α-amylase will allow these chains to be hydrolyzed into smaller fragments called dextrins.The size of the tank is generally designed to achieve a residence time of 1 to 2 hours, so as to allow sufficient contact time for the enzyme to sufficiently degrade the starch chains into dextrins. According to a first variant of the process, the resulting dextrin solution is cooled to 60°C before being transferred to another tank. Another enzyme such as glucoamylase is added to hydrolyze the dextrins into glucose. This operation, called pre-saccharification, typically takes between 3 and 4 hours depending on the dosage of the enzyme and allows for the minimum glucose level necessary for the growth of microorganisms to be obtained. The resulting glucose solution is cooled again to reach a temperature of approximately 30°C and is then transferred to a so-called fermentation tank. In the case of ethanol production, yeasts are added to the tank.These microorganisms will consume the glucose and produce ethanol, intended in particular for the food, pharmaceutical, and green chemistry industries. According to a second variant of the process, no pre-saccharification is carried out but a propagation step. The dextrin solution obtained after liquefaction is directly cooled to 30°C to be injected into a tank. At the same time, the yeast and glucoamylase are injected. Since the temperature is 30°C, the glucoamylase has a reduced activity but is sufficient to provide the glucose necessary for yeast growth. In cases where glucose from wheat is used in the production of organic acids, vitamins, or amino acids, the process for obtaining glucose 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 so that all the dextrins have been converted into glucose. At the end of saccharification, the glucose level must be greater than 95% on a dry matter basis. For these productions of organic acids, vitamins or amino acids, the glucose solution must be freed from 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 steps. When the raw material is beet, the sugar extraction process begins by washing the root to remove the rootlets and then the washed beet is cut into small sticks called cossettes.These are introduced into a device, called a diffuser, containing a volume of water that circulates counter-currently 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 "diffusion juice") comes out at the top of the diffuser and the sugar-depleted cossettes come out at the bottom of the diffuser in the form of pulp. The diffusion juices are then subjected to a lime treatment (or liming) in order to precipitate some of the impurities present in the juices. Liming is generally followed by a double carbonation (addition of CO2) to precipitate the lime remaining in the juice. The impurities and precipitated lime are then separated from the juice by filtration. The purified juice is then subjected to an evaporation step to concentrate it until a syrup with a sugar concentration close to saturation is obtained.Evaporation typically takes place in a "multiple-effect" evaporator (several successive evaporators) in which the pressure is lowered in effect to reduce the boiling point of the concentrated juice. The syrup obtained can then be used in a fermentation process which is most often intended for the production of ethanol. The processes described above are 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 find significant quantities of mineral nitrogen in the soil to grow (2 to 3 kg per 100 kg of corn or wheat).This nitrogen is then provided by nitrogen fertilizers, which are the main cause of greenhouse gas emissions linked 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 fertilizers. There is therefore a real need to develop a process for preparing a substrate that can be used in fermentation, particularly for the production of ethanol or other organic compounds on an industrial scale, with reduced greenhouse gas emissions.Summary of the invention The invention relates firstly to a method for preparing a fermentation substrate, comprising the following steps: – providing at least one legume seed comprising starch and proteins; – 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 water vapor with the starch fluid, so as to obtain a hydrolyzed starch fluid.In embodiments, the method 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-1,6-glucosidase. In embodiments, the method further comprises a step of introducing into the starch fluid at least one enzyme selected from the group consisting of saccharidases, preferably one α-amylase. In embodiments, the legume seed comprises a skin, and the method comprises a step of dehulling the seed prior to micronization. In embodiments, the starch hydrolysis is carried out using a direct steam injection device in continuous mode.In embodiments, the purification of the micronized fraction comprises an aeraulic separation step, preferably by means of a cyclone with selector. In embodiments, the legume is selected from the group of beans, peas, broad beans, lentils, chickpeas and mixtures thereof. In embodiments, the method generates a greenhouse gas emission of less than 100, preferably 60, kg of CO2 oil equivalent per tonne of legume seed used. The invention also relates to a fermentation substrate obtained by a method as described above. The invention also relates to a fermentation method comprising contacting a fermentation substrate as described above with at least one microorganism.The invention also relates to a method for producing a fermentation product, comprising the following steps: – preparing a fermentation substrate according to a method as described above; and – contacting said fermentation substrate with at least one microorganism, so as to obtain a fermentation product. In embodiments, the fermentation product produced by the above method comprises at least one compound selected from the group consisting of alcohols, preferably ethanol, organic acids, amino acids, vitamins and mixtures thereof. In embodiments, the microorganism is selected from the group consisting of yeasts, bacteria and combinations thereof. The invention also relates to a fermentation product obtained by a method as described above.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. The present invention makes it possible to meet the need expressed above. More particularly, it provides a method for preparing a fermentation substrate that is more environmentally friendly, and more particularly with low greenhouse gas emissions, while allowing the production of a substrate rich in nutrients for fermentation, and in particular in glucose, and being capable of being produced on an industrial scale.This is achieved by using a specific raw material, namely a legume, and by combining a step of micronizing said legume with a step of purifying the micronized legume, allowing the production of a fraction enriched in starch and depleted in protein. Brief description of the figures Figure 1 represents a schematic representation of an example of a continuous direct steam injection device usable in the invention. The arrows represent the flow direction of the streams. Detailed description The invention is now described in more detail and in a non-limiting manner in the following description. Unless otherwise indicated, all percentages are mass percentages. In the present text, the quantities indicated for a given species can apply to this species according to all its definitions (as mentioned in the present text), including the more restricted definitions.The invention relates to a method for preparing a substrate from at least one legume seed. The term "legume" means plants of the Fabaceae family. Legume seeds comprise, in particular, starch and proteins. They have the advantage of being rich in carbohydrates (they may comprise approximately 60% carbohydrate), essentially in the form of starch. 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. 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 nitrogen from the air in the soil thanks to their symbiotic association with bacteria of the genus Rhizobium via the formation of nodules, which allows the supply to the plant of the nitrogen necessary for its growth. The capacity of legumes to fix nitrogen from the air makes it possible to avoid the use of nitrogen fertilizers which, applied in excess, harm the biodiversity of the soil and therefore its fertility. In addition, 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 legumes is returned to the next crop via the decomposition of crop residues (aerial and underground parts) by Rhizobium bacteria, the most easily degradable residues (leaves, stems with low woody carbon / nitrogen ratio) decomposing and releasing nitrogen in a few weeks while the woody parts (stems, roots) mineralize more slowly. More specifically, carbon emissions linked to legume cultivation are estimated at 200 kg of CO2 oil equivalent per tonne of legume.When the legume crop is 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 of CO2 oil equivalent per tonne of legume: the legume crop therefore has in this case a net near-neutral balance of 11 kg of CO2 oil equivalent per tonne of legume. Legume seeds are a plant material comprising a skin and a kernel. However, in this text, the term "seed" can generally refer to the whole seed as well as any part of the seed (e.g., the kernel), unless otherwise indicated. Preferably, a legume seed comprising a skin and a kernel is used as the starting raw material.Advantageously, the method according to the invention comprises a step of removing the skin (or dehulling) from the legume seeds. Indeed, the skin is mainly composed of insoluble fibers which are not consumed by the fermentation microorganisms. In addition, the majority of contaminants in the legume seed are found in the skin, its removal thus reduces the risks of contamination of the prepared substrate. In addition, the presence of fibers also increases the viscosity of the prepared fermentation substrate, which limits the dry matter content of this substrate. The mass quantity of fibers can for example represent 8 to 10% of the dry matter of the total legume seed. Preferably, the dehulling is a mechanical dehulling, carried out for example by abrasion, compression, impact, shearing or any other appropriate mechanical action.Advantageously, the dehulling is carried out by grinding the seed and then separating the particles obtained according to their size. 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 pendulum grinder using compression force can be used. At the end of the grinding, a mixture of skin fragments and almond powder (also called flour in the present text) is obtained. The step of separating the particles obtained is preferably carried out by sieving. In particular, the particles can be separated by passing them over a suitable mesh sieve to separate the skin fragments from the flour. For example, particles with a size less than a size of between 200 and 600 mn can be separated from skin fragments of a size greater than or equal to such a size.The legume seed, preferably the hulled seed, more preferably the flour, is subjected to a grinding step and a ground fraction is collected. Particularly advantageously, the grinding comprises, or is, micronization, preferably by dry process. The ground fraction obtained is then a micronized fraction. Legumes have a protein content on dry matter much higher than plants conventionally used as raw material in fermentation industries (cereals, beets and sugar canes). The protein content of legumes can for example reach up to approximately 30% by weight on dry matter compared to approximately 10 to 12% by weight for cereals and even considerably less for beets and sugar cane. Such a high protein content causes certain difficulties in conventional preparation processes.First of all, during the starch hydrolysis step, the applied temperature causes the proteins to coagulate, making them insoluble. A large quantity of insoluble proteins, combined with the presence of fibers, can lead to the formation of a magma that is difficult to transfer from one step to another and creates a risk of creating a plug blocking the passage in the pipes. In addition, the formation of a magma restricts the technologies that can be used during the hydrolysis step, complicating or preventing the use of certain devices, in particular direct steam injection in continuous mode. Indeed, in such devices, the formation of a protein and fiber plug can lead to a very significant increase in pressure in the device and cause damage to the device (in particular the explosion of the seals), or even its explosion, which can also injure an operator nearby.In addition, a large amount 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 unwanted microorganisms such as, for example, acetic acid bacteria instead of yeasts in the case of ethanol production. The micronization step in combination with the subsequent purification step makes it possible to overcome the above-mentioned disadvantages caused by a large amount of protein. The micronization step makes it possible to separate the proteins from the starch granules to allow their subsequent separation. By "micronization" is meant a grinding process allowing the production of particles with a volume median diameter of less than 100 µm, preferably less than 50 µm, and even more preferably less than 30 µm. The volume median diameter (D50) of the particles can be measured according to the NF ISO 13320-1 standard.Micronization can be carried out by any suitable grinder (in particular, any grinder using mechanical forces of abrasion, compression, impact or shear). For example, a grinder using impact force can be used. Micronization is very preferably carried out at room temperature (i.e. between 15 and 30 °C). Micronization has the additional advantage of being low greenhouse gas emitting, it is preferably carried out at room temperature and by a dry process. The method according to the invention comprises a step of purifying the micronized fraction. Advantageously, this purification comprises a step of separating the starch granules from the proteins. At the end of the purification, a fraction enriched in starch and depleted in protein is collected. A fraction enriched in protein and depleted in starch is also preferably recovered.By "fraction enriched in starch and depleted in protein" is meant a fraction in which the ratio of molar proportions of starch / protein (on dry matter) is higher than that of the fraction subjected to purification. By "fraction enriched in protein and depleted in starch" is meant a fraction in which the ratio of molar proportions of starch / protein (on dry matter) is lower than that of the fraction subjected to purification. Given the difference in size between proteins and starch granules (D50 of about 1 to 5 µm for proteins and about 10 to 30 µm for starch granules) and in density, a separation based on a difference in size, density or weight of the particles can advantageously be used. Preferably, the separation is an aeraulic separation.By "aeraulic separation" is meant any separation technology using a jet of gas (preferably air) entraining at least some of the particles to be separated. More preferably, the separation is a cyclonic separation. It can be carried out by means of a cyclone, advantageously associated with a selector. By "selector" is meant any variable-speed rotating element equipped with radial blades installed in a part (preferably the upper part) of a cyclonic separator. This equipment makes it possible to increase the separation efficiency of the particles according to their density. The use of an aeraulic separation device allows the recovery of the lightest particles, entrained by the gas flow, at one end of the device (fraction enriched in protein and depleted in starch) while the heavier particles are collected at another end (fraction enriched in starch and depleted in protein).Preferably, the starch-enriched and protein-depleted fraction comprises a quantity of 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, more preferably 60 to 80% by weight (relative to the total weight of dry matter of the fraction). In particular, the quantity of carbohydrates in the recovered starch-enriched and protein-depleted 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 weight of dry matter of the fraction.Preferably, the starch-enriched and protein-depleted fraction comprises a protein amount of less than or equal to 30% by weight, preferably an amount of 0.5 to 30% by weight, more preferably 3 to 20% by weight, more preferably 5 to 15% by weight (based on the total dry matter weight of the fraction). In embodiments, the protein amount in the recovered starch-enriched and protein-depleted fraction may be, based on the total dry matter 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.Preferably, the starch-enriched and protein-depleted fraction comprises a quantity of fiber less than or equal to 10% by weight, preferably an amount of 0.5 to 10% by weight, more preferably 1 to 6% by weight, relative to the total weight of dry matter of the fraction; in particular the fraction may comprise a quantity of fiber of 0.5 to 2% by weight, or 2 to 4% by weight, or 4 to 6% by weight, or 6 to 8% by weight, or 8 to 10% by weight, relative to the total weight of dry matter of the fraction. The term "fiber" means all plant polymeric molecules, soluble or insoluble, other than starch and starch fragments. Fibers include in particular cellulose, hemicellulose, lignin, β-glucans and pectin.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 weight of dry matter 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 weight of dry matter of the fraction. According to the method 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 comprising starch). The liquid is preferably water. The starch fluid is preferably in the form of a dispersion, more preferably it is a starch milk.By "starch milk" is meant a suspension of starch in water (said suspension may comprise other components, solubilized in water or not). Preferably, 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. Very advantageously, at least one enzyme is introduced into the starch fluid (preferably the starch milk). The enzyme is preferably a saccharidase, and more particularly an α-amylase. Most preferably, the enzyme is thermostable, in particular 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 starch dry matter, preferably 0.3 to 0.5 kg of enzyme per tonne of starch dry matter. The pH of the starch fluid 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 efficiency of the enzyme. α-amylases are enzymes capable of hydrolyzing starch into dextrins. However, at this stage of the process, the α-amylases do not have access to the starch molecules which are enclosed in granules. 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, so as to allow the action of the enzymes (this step of causing the starch granules to burst can also be called "liquefaction").The bursting of the starch granules is carried out by heating the starch fluid (preferably starch milk) to a temperature that allows the introduction of liquid into the granule, thus causing the granule to swell and then burst. A hydrolyzed starch fluid is then obtained. According to the invention, the hydrolysis is carried out by mixing water vapor with the starch fluid, preferably using a direct steam injection device. Preferably, the starch fluid is heated (by mixing with the water vapor) to a temperature (referred to in the present text as the hydrolysis temperature or liquefaction temperature) of 90 to 130°C, preferably 90 to 100°C. Particularly advantageously, the hydrolysis is carried out by mixing a stream of water vapor with a stream of the starch fluid. The term "stream" means a fluid (gas or liquid) in motion.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 the two fluids, into the mixer is carried out at least in part simultaneously with the discharge of said mixture from the mixer. The mixing of water vapor with the starch fluid in the form of a flow allows a very rapid, even almost instantaneous, rise in temperature of the starch fluid. Compared to the use of tanks (generally 300 to 500 m. 3, for a residence time of 1 to 2 hours) equipped with steam injection rods which are conventionally used, the mixing of fluids in the form of a stream in continuous mode allows for faster heating, a reduction in steam consumption, and a reduction in energy consumption. It is estimated that greenhouse gas emissions can be reduced by approximately 40%. Therefore, implementing the hydrolysis step by continuously mixing the water vapor with the starch fluid in the form of a stream allows for an even greater reduction in the greenhouse gas emissions of the process. In a particularly preferred manner, the hydrolysis is carried out using a direct steam injection device in continuous mode, more preferably a "jet-cooker" device. An example of such a device is shown in Figure 1. Preferably, the direct steam injection device in continuous mode comprises a starch fluid supply line1 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 injection points of the steam and the starch fluid into the mixing tube 3 are arranged in a coaxial arrangement. Advantageously, the steam injector 4 forms a needle in the starch fluid supply line 1. The flows of starch fluid and steam are mixed within the mixing tube 3. The mixing tube 3 comprises a fluid outlet 5, connected to an outlet pipe allowing the discharge of the mixture. The flows of starch fluid and steam can be adjusted independently of each other, for example by means of valves. 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 may be0.2 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 water vapor injected into the mixing tube is advantageously 0.4 to 1.5 Mpa, more preferably 0.6 to 1 Mpa. In particular, the pressure of the injected water vapor may 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, more preferably 0.2 to 0.4 Mpa. In particular, the pressure of the mixture at the outlet of the mixing tube may 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 may for example be 0.1 to 0.2 Mpa, or 0.2to 0.3 Mpa, or from 0.3 to 0.4 Mpa, or from 0.4 to 0.5 Mpa. Such pressure ranges allow optimal homogenization of the water vapor / starch fluid mixture. 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 D1 worth 1.5 to 2.5 times the diameter DN over a pipe length of 10 to 30 times the diameter DN, then – a diameter D2 worth 2 to 5 times the diameter DN. Such a geometry makes it possible to control the increase 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. The hydrolyzed starch fluid, preferably not cooled, 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 to hydrolyzestarch. 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 transformed into glucose. The method used for measuring the DE is the Lane-Eynon method. The hydrolyzed starch fluid can be used as a fermentation substrate, optionally after one or more additional treatments. The method according to the invention can in particular comprise a step of introducing at least one enzyme into the hydrolyzed starch fluid. This step is called "saccharification" and allows the hydrolysis of dextrins into glucose. In the present text, the term "saccharification" is used to designate any process of hydrolysis of dextrins into glucose, regardless of the degree of hydrolysis achieved; The saccharification step can also be called "pre-saccharification" when hydrolysis is notcomplete or almost complete. Preferably, prior to the introduction of the enzymes, the hydrolyzed starch fluid is introduced into a tank. The enzyme(s) introduced are preferably chosen from the group consisting of glucosidases. More preferably, at least 2 enzymes are introduced into the hydrolyzed starch liquid, more preferably at least one α-1,4-glucosidase and one amylo-α-1,6-glucosidase are introduced into the hydrolyzed starch liquid. The α-1,4-glucosidase hydrolyzes the α-(1,4) bonds involved in the linear glucose chains of dextrins; the amylo-α-1,6-glucosidase enzyme (also called "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 set between 50 and 70°C, preferably between 55 and 65°C. These conditions allow optimal functioning of the enzymes.The quantity of enzymes introduced may be from 0.2 to 0.6 kg per tonne of dry starch matter. 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"). In other embodiments, the duration of the saccharification is from 30 to 70 hours, preferably from 40 to 60 hours. Advantageously, the quantity 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 quantity of glucose is determined according to standard NF EN ISO 10504. A glucose-enriched medium is obtained, which can be used as a fermentation substrate, 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 ishigher than that of the hydrolyzed fluid before saccharification. Preferably, when the substrate undergoes a pre-saccharification step of 2 to 6 hours, it is not subjected to further purification. Preferably, when the substrate undergoes a saccharification step of 30 to 70 hours, it undergoes at least one subsequent purification step, preferably filtration and demineralization, preferably using ion exchange resins. Advantageously, the method 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 method for preparing a fermentation substrate according to the invention can generate ...).greenhouse from 0 to 20, or from 20 to 30, or from 30 to 40, or from 40 to 50, or from 50 to 60, or from 60 to 80, or from 80 to 100, kg of CO2eq per tonne of legume seed used). The greenhouse gas emissions can be determined as indicated in the Examples section below. The invention also relates to a fermentation substrate obtained by, or obtainable 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 it is obtained by a process including a pre-saccharification step): – an amount of protein, relative to the total weight of dry matter of the substrate, of 5 to 25% by weight, preferably of 15 to 20% by weight, for example of 5 to 10% by weight, or of 10 to 15% by weight, or of 15 to 20% by weight, or of 20 to 25% by weight; – an amount of carbohydrate, relative to the total weight of dry matter of the substrate, of 40at 80% by weight, preferably from 60 to 75% by weight, for example from 40 to 50% by weight, or from 50 to 60% by weight, or from 60 to 70% by weight, or from 70 to 80% by weight; – an amount of fat (lipids), relative to the total weight of dry matter of the substrate, of 0.5 to 5% by weight, preferably from 0.5 to 3% by weight, for example from 0.5 to 1% by weight, or from 1 to 2% by weight, or from 2 to 3% by weight, or from 3 to 4% by weight, or from 4 to 5% by weight; – an amount of fiber, relative to the total weight of dry matter 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 can advantageously comprise one or more of the following characteristics (in particular when it is obtained by aprocess including a saccharification step of 30 to 70 h): – an amount of carbohydrate, relative to the total weight of dry matter of the substrate, greater than or equal to 90% by weight, preferably greater than or equal to 95% by weight; – an amount of protein, relative to the total weight of dry matter 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 weight of dry matter 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 bringing a fermentation substrate as described above into contact with at least one microorganism. The microorganism is preferably chosenfrom the group consisting of yeasts, bacteria and combinations thereof. In embodiments, the microorganism is contacted with, as a substrate, a glucose-enriched medium (i.e., having undergone the saccharification step) as described above. For this, 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, even more preferably 26 to 30°C prior to its contact with the microorganism. In other embodiments, the microorganism, preferably one or more yeasts, is contacted with, as a substrate, a hydrolyzed starch fluid (i.e., not having undergone the saccharification step) as described above. Preferably, the hydrolyzed starch fluid is precooled to a temperature of 20 to 40°C, preferablystill from 25 to 35°C, more preferably from 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 chosen from the group consisting of glucosidases. Particularly preferably, the substrate is brought into contact with the microorganism and with at least one α-1,4-glucosidase and one amylo-α-1,6-glucosidase. This step is called the “propagation step”. Advantageously, the substrate is introduced into a tank and the microorganism and the enzymes are added to the tank. The amounts of enzyme and the pH are advantageously as described above in relation to the saccharification step. The invention also relates to a fermentation process, or a process for producing a fermentation product, comprising the steps of: – preparing a fermentation substrate according to the process described above; and – bringing the substrate into contact withfermentation with at least one microorganism. The step of bringing the fermentation substrate into contact with at least one microorganism may be as described above. It corresponds to a fermentation step. Bringing the fermentation substrate into contact with the microorganism(s) advantageously results in obtaining a fermentation product, in particular as described below. The invention also relates to a fermentation product obtained by, or capable of being obtained by, a fermentation process, or for producing a fermentation product, 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. Preferably, when the fermentation product is or comprises a compound selected from organic acids, amino acids, vitamins andmixtures thereof, a substrate having been prepared by a process comprising a saccharification step of 30 to 70 hours is used in the fermentation process (or in the production of a fermentation product). Preferably, when the fermentation product is or comprises a compound chosen from alcohols, and is in particular ethanol, a substrate having been prepared by a process comprising a pre-saccharification step is used in the fermentation process (or in the production of a fermentation product) or a hydrolyzed starch fluid is used as substrate (in the latter case, the fermentation process or in the production of a fermentation product very preferably comprises a propagation step as described above). Examples The following examples illustrate the invention without limiting it. A process for producing ethanol by fermentation using a substrate prepared from a legume (pea) according to the invention was compared to aethanol production process by fermentation using a substrate prepared from cereal (wheat) and a process for producing ethanol by fermentation using a substrate prepared from beet. Ethanol production process from beet 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. Once out of the washhouse, the beets are cut into cossettes (whose shape is reminiscent of fries). The size of the cossettes is 5 to 6 cm long and 2 to 3 mm wide. This cutting is done using a root cutter type Model 2000 – 600 – 60 from the company MAGUIN. The cossettes are then conveyed to the diffusion, in which they circulate counter-currently with hot water at 80°C, in which the soluble compounds of the beet migrate to give a diffusion juice exiting at the top ofdiffuser. The cossettes come out of the diffuser tail in the form of pulps and some of the water they contain is removed by pressing or dehydration for recycling. The pulps can then be used in animal feed. The diffusion juice then undergoes purification by a quicklime treatment resulting in the precipitation of some of the impurities. Quicklime is obtained by calcining limestone at 900°C at a rate of 4.6 kg of stone per ton 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 precipitated lime are separated from the juice by filtration using a filter press. The purified juice contains 85% water by weight. It is subjected to evaporation in order to be concentrated, until a syrup is obtained with a sucrose concentration close to saturation, i.e. 60 to 70% by weight.Evaporation takes place in a multiple-effect evaporator, the pressure being lowered in effect to reduce the boiling temperature of the concentrated juice, which prevents it from being cooked. Concentrating the juice prevents sugar fermentation and thus allows the juice to be stored before it is sent to the distillery workshop. The fermentation stage begins with the preparation of the pre-fermentation. The syrup is diluted to 7% by weight of sugar (sucrose) by adding water and is then introduced into a tank called a pre-fermenter with a solution of Saccharomyces cerevisiae yeasts. The quantity of yeast is 6 g per tonne of sugar. Urea, at a rate of 1.3 kg per tonne 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.When the alcohol content (especially ethanol) has reached 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 of between 20 and 25% (strong must) is added in a proportion of 30 to 50% by weight of the weak must. The alcoholic fermentation, which is exothermic, continues while the temperature is maintained between 30 and 35°C by means of a plate heat exchanger which is itself connected to a cooling tower. Fermentation takes place for a period of between 30 and 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 has been converted into ethanol and carbon dioxide, the alcohol content in the fermenter is between 10 and 14% by weight. The fermentation must is then subjected to a distillation stageconsisting 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. 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 stage: – 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 veryvolatile and poorly soluble in water are carried to the top of the column, while the water-soluble ethanol and methanol are carried to the bottom of the column. – Rectification column: The alcohol-water mixture from the extraction column is distilled again until it reaches almost the azeotropic alcohol / water mixture (97% by weight of alcohol) at the bottom of the column while the impurities will be collected at the top of the column (distillate). The impurities recovered from the previous column are introduced into this distillate. – Demethylation column: This large column containing a very large number of trays allows the separation of methanol and other impurities from the ethanol. The product obtained is called "superfine alcohol". The residue containing the methanol is mixed with the impurities collected from the previous step. – Head column: All the impurities from the previous columns are passed through this column in order to purify them to producefusel oils. The ethanol recovered at the bottom of the column is recycled into the extraction column or the rectification column to improve the ethanol purification yield. The fusel oils are collected at the top of the column. All rectification operations use the liquid separation process by vaporization-condensation fractionation. During the distillation step, a non-soluble residue is collected with the unevaporated water. This residue is called vinasse. The dry matter is approximately 6% by weight. Since this dry matter is too low, the residue is introduced into a falling-flow tubular evaporator. Evaporation is carried out under vacuum (0.02 MPa pressure) with steam injection. The final dry matter of the residue is 30 to 35% by weight. Concentrated vinasse can be used in agriculture, particularly for the preparation of an amendment. For an ethanol production unit, the quantity of CO2 oil equivalent (CO2eq)has been estimated. The quantity of oil equivalent CO2 represents all greenhouse gas emissions (also 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 ERNÄHRUNG (BLE): Leitfaden Nachhaltige Biomasseherstellung, 1 èreEdition, Bonn, 2010; – lINAS INTERNATIONALES INSTITUT FÜR NACHHALTIGKEITSANALYSEN UND -STRATEGIEN, ÖKO- INSTITUT EV INSTITUT FÜR ANGEWANDTE ÖKOLOGY EV, Gemis, 2014. The results for the process of ethanol production from a beet substrate described above are presented in the table below. The ethanol production is 88830 t / year (from 1189256 t of beet). The CO2eq emitted during sugar extraction is attributed at a rate of 94% by weight to the production and processing of the sugar juice and at a rate of 6% by weight to the production of the pulp (which is indicated under the heading "yield considered" in the table below). [Table 1] The total amount of CO2eq calculated for the entire process is distributed 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 vinasse co-product. The share of greenhouse gas emissions corresponding to ethanol production alone is shown in the table above in the line "Total (kg CO 2eq / t ethanol) without vinasse”. Process for producing ethanol from wheat. The wheat grains are harvested. Unlike beetroot, the 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. The cleaned wheat is then ground using a roller mill, then sieved to separate the bran (wheat skin) from the flour (almond powder). These steps up to the collection of the flour are carried out dry and at room temperature. The flour is then suspended in water so as to have a dry matter content of between 25 and 35%. The fluid thus obtained is introduced into an 80 m tank 3equipped with steam injection rods. At the same time, 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 rods until the fluid reaches a temperature of between 90 and 95°C. This temperature allows water to penetrate the starch granules until the protective membrane of the granules bursts. 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 cut into dextrin chains. The hydrolyzed must is sent to a 50 m stirred tank 3and 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. The following steps (fermentation, distillation and rectification) are identical to those described above for the process of producing ethanol from beet. The quantity of oil equivalent CO2 emitted by the process was estimated as indicated above and the results are presented in the table below. The ethanol production is 23887 t / year (from 80235 t of wheat). The CO2eq emitted up to the saccharification step (not included) is attributed 84% to the production and processing of flour and 16% to the production of fiber (bran) (which is indicated under the heading "yield considered" in the table below). [Table 2] The total amount of CO2eq calculated for the entire process is distributed 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 vinasse co-product. The share of greenhouse gas emissions corresponding to ethanol production only is indicated in the table above in the line "Total (kg CO2eq / t ethanol) without vinasse". Process for producing ethanol from peas. Once harvested, the peas are dehulled by grinding using a pendulum mill using compression force, then the particles produced are passed through a sieve to separate the skins with a size less than 500 µm from the skins with a size greater than or equal to 500 µm.The selected particles (less than 500 µm) are then subjected to a micronization step using a mill using impact force (so as to achieve a median diameter in volume (D50) of the particles less than or equal to 30 µm). At the outlet of the mill, the flour obtained is introduced into turbo-cyclones with selector fed by an air flow to activate a cyclonic effect, in order to separate the particles according to their density. The light particles (called protein fraction), having a D50 of 1 to 5 µm, are carried to the top of the cyclone while the heavier particles, having a D50 of 10 to 30 µm, are carried to the bottom. The heavy fraction (called starch fraction) is recovered and mixed with water so as to achieve a fluid having a mass content of dry matter of approximately 30%.The pH is adjusted between 4.0 and 6.0 by sulfuric acid and then an alpha amylase enzyme (maxamyl HT) is added to the fluid in a quantity of 0.3 to 0.5 kg per tonne of dry starch matter. In order to cause the starch granules it contains to burst, the fluid is then heated to 100°C by direct injection of steam in continuous mode in a Hydro-thermal K510 type jet-cooker (pressure of the fluid including the starch granules: 0.6 MPa; pressure of the water vapor: 0.9 MPa; pressure of the fluid / steam mixture at the jet-cooker outlet: 0.25 MPa). The fluid is then subjected to a dextrinization step: for this, the uncooled fluid is placed in a 30 m tank. 3for a period of 4 hours The fluid cooled to 60°C is then introduced into another tank to undergo a pre-saccharification step. An α-1,4-glucosidase and an amylo-α-1,6-glucosidase (Deltazyme GA LE-5) are introduced into the fluid in an amount of 0.3 kg per tonne of dry starch matter, and the pH is adjusted to a value between 4.0 and 4.5 by sulfuric acid. The fluid is left in the tank for a period of 3 hours. The following 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 fumes from the evaporator used to concentrate the vinasse, recompressed using a mechanical re-compressor. The energy used in this case is electricity and not gas.The amount of oil equivalent CO2 emitted by the process was estimated as indicated above and the results are presented in the table below. The ethanol production is 7465 t / year (from 32982 t of wheat). The CO2eq emitted up to the pre-saccharification stage (not included) is attributed at a rate of 67.2% by weight to the production and processing of the starch fraction, at a rate of 24% by weight to the production of the protein fraction, and at a rate of 8.8% to the production of fibers (film) (which is indicated under the heading "yield considered" in the table below). [Table 3]. The total amount of CO2eq calculated for the entire process is distributed 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 vinasse co-product. The share of greenhouse gas emissions corresponding to ethanol production only is shown in the table above in the row "Total (kg CO2eq / t ethanol) without vinasse". The results are summarized in the table below (the shares of emissions attributed to ethanol production only are considered): [Table 4] It is found that the ethanol production process according to the invention emits significantly fewer greenhouse gases than comparative processes using a substrate prepared from cereals and beetroot. Furthermore, with regard to the process up to distillation, the process according to the invention produces very low greenhouse gas emissions.

Claims

Claims 1. A method for preparing a fermentation substrate, comprising the following steps: – providing at least one legume seed comprising starch and proteins; – 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 water vapor with the starch fluid, so as to obtain a hydrolyzed starch fluid. 2.The method of 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 α-1,4-glucosidase and one amylo-α-1,6-glucosidase.

3. The method of 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 one α-amylase.

4. The method of one of claims 1 to 3, wherein the legume seed comprises a skin, and wherein the method comprises a step of dehulling the seed prior to micronization.

5. Method according to one of claims 1 to 4, in which the purification of the micronized fraction comprises a step of. aeraulic separation, preferably by means of a cyclone with selector.

6. Method according to one of claims 1 to 5, in which the hydrolysis of the starch is carried out using a direct steam injection device in continuous mode.

7. Method according to one of claims 1 to 6, in which the legume is chosen from the group of beans, peas, broad beans, lentils, chickpeas and mixtures thereof.

8. Method according to one of claims 1 to 7, generating a greenhouse gas emission of less than 100 kg of CO2 oil equivalent per tonne of legume seed used, preferably 60 kg of CO2 oil equivalent per tonne of legume seed used.

9. Fermentation substrate obtained by a method according to one of claims 1 to 8.

10. Fermentation method comprising bringing a fermentation substrate according to claim 9 into contact with at least one microorganism. 11.A method of producing a fermentation product, comprising the following steps: – preparing a fermentation substrate according to the method according to one of claims 1 to 8; and – bringing said fermentation substrate into contact with at least one microorganism, so as to obtain a fermentation product.

12. A method according to claim 11, wherein 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.

13. A method according to one of claims 10 to 12, wherein the microorganism is selected from the group consisting of yeasts, bacteria and combinations thereof.

14. Fermentation product obtained by a method according to one of claims 10 to 13.

15. Fermentation product according to claim 14, comprising at least one compound selected from the group consisting of alcohols, preferably ethanol, organic acids, amino acids, vitamins and mixtures thereof.

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