Method for utilizing by-products from agricultural food industry

Abstract

The invention relates to a method for utilizing by-products of edible flour processing, in which a purified solution containing monosaccharides is obtained by hydrolysis of the starch component of the waste and solid residues rich in insoluble fibers and proteins and / or oligopeptides and liquid fractions rich in soluble proteins and fibers are separated.

Description

[0001] The present invention relates to a method that uses byproducts from the agri-food industry, such as waste biomass from edible flour processing, as raw materials to obtain a variety of high-value-added organic compounds that can be utilized through reuse in the development of industrial applications.

[0002] In fact, this method enables the extraction, separation, and recovery of second-generation sugars suitable for chemical and fermentation processes from the structural carbohydrates present in these biomass, while also optimizing the extraction of fiber and protein, which can be widely used in animal feed or the food industry.

[0003] The sugar known as "second-generation" sugar is obtained from biomass waste from the agricultural and food industries, and therefore does not consume human food resources.

[0004] Through various chemical, physical / chemical and / or enzymatic treatments, these biomasses can actually be broken down into their main components (starch, cellulose, hemicellulose, lignin and protein), and monosaccharides can be obtained from the polysaccharide-rich components for use as raw materials to produce chemical compounds for the development of end applications (such as fuels like ethanol, polymers like PHA, monomers like 1,4-butanediol, etc.).

[0005] The quality and composition of second-generation sugars determine how these sugars can be used as carbon sources in culture media for the growth and production of various microorganisms such as bacteria, fungi, or yeasts (possibly genetically modified) during fermentation.

[0006] In fermentation, the culture medium serves as the medium for microbial growth, thus providing the necessary elements for normal growth and the production of metabolites. For example, carbon is a major element in cell composition and is also used by compounds as an energy source. However, the ability of microorganisms to utilize certain carbon compounds varies by species and strain. Some microorganisms preferentially consume simple sugars containing six carbon atoms, such as glucose and fructose; this is the case, for example, with microorganisms used to produce biobutanediol. Disaccharides can be metabolized by a smaller number of microorganisms; for example, see patent application WO2015 / 158716, which describes a method for producing 1,4-butanediol comprising fermentation in a culture medium containing a mixture of glucose and sucrose by microorganisms having at least one metabolic pathway for the synthesis of 1,4-butanediol. In contrast, complex pentoses and sugars are used by only a small number of microorganisms.

[0007] Furthermore, in the specific cases of cereal (including false cereals) and legume crops, the milling industry, which is well known to involve the production of edible flour as the main product, produces intermediate and by-products from various stages of processing. These products contain starch, salt, soluble fiber (glucan, inulin, some hemicellulose, etc.), insoluble fiber (cellulose, hemicellulose, and lignin), oil and fatty acids, as well as organic nitrogen in the form of proteins, oligopeptides, or simple amino acids. These intermediates and secondary products, rich in protein and / or fiber, are therefore suitable for use as ingredients and / or semi-finished products in food and nutritional supplements.

[0008] However, they do require further processing steps to remove unwanted components such as residual starch, salt, oil, and fatty acids, the amounts of which vary depending on the starting product (cereals, seeds, legumes) and the degree and type of milling.

[0009] On the other hand, the application of second-generation sugars in fermentation also requires a sufficiently high level of purity to avoid inhibiting microbial growth and interfering with the fermentation process and the purification of the resulting compounds. It is well known that impurities generated during the production of second-generation sugars, such as organic acids, high molecular weight molecules, and high concentrations of salt, can adversely affect microbial metabolism and impair their application in the fermentation process.

[0010] At the industrial level, fermentation processes typically require substrates with limited impurity content at appropriate concentrations so that they can be used without interfering with production and / or purification processes.

[0011] For example, commercially available monosaccharide syrups, obtained directly from corn or potato starch, typically have a nitrogen and ash content of less than 0.05% by weight.

[0012] This invention overcomes the aforementioned drawbacks by enabling the full utilization of byproducts from edible flour processing. In fact, the method according to the invention can completely convert existing starch into a highly purified and concentrated monosaccharide syrup, which is therefore suitable for fermentation processes and can be obtained in high yields due to the possibility that other structural carbohydrates (cellulose and hemicellulose) can also be converted into monosaccharides. Simultaneously, the method according to the invention maximizes the recovery of fiber and organic nitrogen compounds (e.g., amino acids, oligopeptides, proteins, etc.) due to the reduction of salt and soluble components, resulting in improved properties and composition. Therefore, all the product of this method is suitable for high-value-added new applications in both the chemical and food industries.

[0013] In particular, the object of the present invention is to provide a method for utilizing a byproduct from the processing of edible flour containing starch, the method comprising the following steps:

[0014] (a) The byproduct is subjected to starch hydrolysis in the presence of water and preferably one or more enzymes to obtain an aqueous mixture comprising monosaccharides, fiber, protein and / or oligopeptides and optionally salts, oils and fatty acids.

[0015] (b) The aqueous mixture is subjected to solid / liquid separation to obtain a solid fraction containing insoluble fiber and insoluble protein and / or oligopeptides, and a liquid fraction containing soluble monosaccharides, fiber and soluble protein and / or oligopeptides and optional salts.

[0016] (c) Purifying the liquid fraction from step b) includes:

[0017] (c1) At least one membrane separation operation to remove the soluble protein fraction and obtain an aqueous solution containing soluble monosaccharides; and subsequently

[0018] (c2) Pass the aqueous solution through a bed containing one or more ion exchange resins at least once to obtain a purified solution containing monosaccharides.

[0019] In the context of this invention, "byproducts of edible flour processing" refers to biomass obtained as a byproduct of milling cereals, pseudo-cereals, legumes, or mixtures thereof, which contains starch and other structural carbohydrates such as cellulose and / or hemicellulose.

[0020] Compared to the starting biomass, the solid fraction obtained in step b) is substantially starch-free and rich in fiber and organic nitrogen (in the form of proteins and / or oligopeptides). The solid fraction can be advantageously further separated into protein and fiber-rich components.

[0021] According to one aspect of the invention, the method optionally includes the step of drying the solid fraction separated in step b) to facilitate the subsequent separation of the protein component from the fiber-rich component.

[0022] According to one aspect of the invention, step c) of the method optionally includes step c3) concentrating the purified solution containing monosaccharides obtained in step c2).

[0023] Finally, the method according to the invention includes the optional step of growing a microbial strain capable of producing chemical intermediates and / or polyhydroxyalkanoates in the presence of a carbon source comprising monosaccharides from the purified aqueous solution obtained in step c2). This growth step is preferably performed after the concentration step c3) described above.

[0024] Advantageously, steps a)-c) are carried out at a temperature not exceeding 100°C, preferably below 80°C and even more preferably below 60°C, thereby maintaining the structure and function of the components present.

[0025] Monosaccharides produced by the method according to the present invention are, for example, glucose, fructose, arabinose, rhamnose, galactose, mannose, and xylose. The term "polysaccharide" is understood to include, for example, starch, cellulose, and heteropolysaccharides such as hemicellulose (which includes xylan, glucuronyl xylan, arabinoxylan, glucomannan, and xyloglucan).

[0026] The term "oligosaccharide" is used in this application to refer to all molecules that are carbohydrate in nature and consist of two to ten monosaccharide units. Therefore, examples of oligosaccharides include disaccharides such as sucrose, maltose, and cellobiose, as well as some maltodextrins.

[0027] The following will refer to Figure 1 The block diagram in the figure describes the method in more detail.

[0028] The by-product of edible flour processing containing starch added in step a) of the method is derived from one or more cereals selected from cereals or wheat, rice, barley, oats, spelt wheat, rye, millet, and corn; derived from one or more false cereals selected from amaranth, buckwheat, quinoa, and chia seeds; derived from one or more legumes selected from peas, chickpeas, lentils, and broad beans; or derived from mixtures thereof.

[0029] Biomass obtained as a byproduct from the milling of the aforementioned plant species mainly consists of fiber (cellulose, hemicellulose, and lignin), vitamins, proteins, enzymes, mineral salts, and lipids, and contains residual starch, the content of which varies depending on the type and steps of processing from which they originated.

[0030] The starch content of the biomass is particularly preferably less than 70% by weight, more preferably less than 60% by weight, even more preferably less than 50% by weight, on a dry weight basis in the case of cereals and false cereals; and preferably less than 40% by weight, even more preferably less than 30% by weight, on a dry weight basis in the case of legumes. The starch content of the biomass relative to the dry weight of the biomass is preferably greater than 5% by weight, 10% by weight, 15% by weight, or 20% by weight.

[0031] The total fiber content (soluble and insoluble, excluding starch) of the biomass is greater than 20% by weight; particularly preferred is a fiber content of 25% or more, 30% or more, or 40% or more of the dry weight of the biomass.

[0032] The protein content of the biomass is preferably 15% by weight or more, more preferably 20% by weight or more.

[0033] The lipid content of these biomass is preferably less than 5% by weight, more preferably less than 2% by weight, and even more preferably less than 1.5% by weight (based on dry weight); in a particularly advantageous aspect, the lipid content is less than or equal to 1% by weight or 0.5% by weight relative to dry weight.

[0034] The use of cereal biomass, especially wheat, is particularly advantageous.

[0035] As is well known, the purpose of the cereal milling process is to separate the endosperm (which constitutes the starchy and dominant part of the caryopsis) from the bran and germ, thereby obtaining flour (which can be flour or semolina depending on the particle size) from the endosperm. This process typically involves alternating stages of milling (including opening or breaking the caryopsis to reduce the particle size of the endosperm) and separating the flour from their respective bran fractions (i.e., bran, which may contain the germ).

[0036] One or more dehulling stages may be performed before milling, which consist of removing the outermost layer of the caryopsis by abrasive action that can be more or less intense, usually without causing significant damage to the endosperm-rich core used for milling.

[0037] Therefore, the bran and / or germ fractions separated during the milling process constitute byproducts suitable for feeding into the processing of edible flour according to step a) of the method according to the present invention.

[0038] According to a preferred aspect, these byproducts of edible flour processing consist of bran (which may be a finer variant called middlings) that optionally includes the germ, i.e., a portion of the caryopsis remaining after the endosperm has been removed.

[0039] According to another preferred aspect, the byproducts of these edible flour processing include medium bran, fine medium bran, and groats, i.e., products with a higher starch content than bran but still unsuitable for use in the bread-making process. These can be used as is or mixed with bran and / or medium bran.

[0040] Outer and inner bran, for example, contain 15% to 30% starch, 15% to 30% protein and oligopeptides, 20% to 30% hemicellulose, 10% to 15% cellulose, less than 10% lignin, and less than 10% ash relative to the dry weight of biomass.

[0041] The amount of protein and oligopeptide present can be determined by multiplying the present organic nitrogen by an appropriate correction factor.

[0042] On the other hand, fine, medium, and coarse wheat flour typically contain 50% to 80% by weight of starch, 10% to 30% by weight of protein, and 3% to 10% by weight of hemicellulose, relative to the dry weight of biomass.

[0043] The method of the present invention may optionally include a preliminary step of treating the biomass (whether obtained as a byproduct of cereal, pseudo-cereal, and / or legume milling industries) to bring it closer to the hydrolysis carried out in the first step of the method and to make it biologically stable. For example, this preliminary stage or pretreatment advantageously includes heat treatment and / or washing with water, carried out under temperature and time conditions known to those skilled in the art. Other useful preliminary operations may aim to reduce the lipid content or fiber content of the biomass, for example, through hydrolysis mediated by enzymes such as cellulase and hemicellulase.

[0044] According to a preferred embodiment of the invention, the method of the invention therefore includes, prior to step a), a defatting step on the byproduct generated from the processing of starch-containing edible flour, for example by extracting the lipid components in one or more organic solvents or supercritical fluids (e.g., CO2) or by physical extraction.

[0045] Suitable organic solvents include, for example, acetone, ethanol, butanol, hexane, tetrahydrofuran, 2-methyltetrahydrofuran (2-MeTHF), cyclopentylmethyl ether, and cyclopentanone. Among these, ethanol and hexane are preferred; even more preferred is ethanol.

[0046] Physical extraction can be advantageously performed by means of cold pressure, instantaneous controlled pressure drop (DIC), pulsed electric field (PEF), and microwave radiation (MW).

[0047] Solvent-free extraction or green solvent extraction is preferred.

[0048] According to a preferred aspect, the biomass undergoing step a) is in pellet form and contains less than 50% by weight, preferably between 42% and 22% by weight, of dust. Advantageously, its water content is about 10% by weight.

[0049] During step a) of the method according to the invention, the biomass is subjected to hydrolysis (or saccharification) of the polysaccharide chains of starch by one or more enzymes belonging to the hydrolytic enzyme family and suitable for hydrolyzing starch, such as amylase, to obtain monosaccharides. Other hydrolytic enzymes, such as cellulase and hemicellulase, may be used before, during, or during the subsequent treatment of the solid residue separated in step b) to maximize the conversion to monosaccharides and also to hydrolyze the polysaccharide chains of cellulose and hemicellulase. Amylase and cellulase are preferred.

[0050] These enzymes can be used alone or in combination, and can be added to the starting biomass at different times depending on the desired hydrolysis products, with appropriate operating conditions for each enzyme.

[0051] Depending on the enzyme selected, those skilled in the art will be able to employ the necessary hydrolysis or saccharification conditions (reaction medium, pH, temperature, duration, etc.). Enzymes that enable the production of a mixture of monosaccharides primarily containing glucose are particularly suitable for use in step a) of this method.

[0052] These enzymes are preferably amylases and selected from α-amylase, glucosylamylase (or amyloglucosidase or γ-amylase) and mixtures thereof. Examples of suitable commercial products are “Spezyme Alpha PF” and “Optidex L-400” provided by IFF.

[0053] It is preferred to use at least one α-amylase and at least one glucosylase or a mixture thereof.

[0054] The hydrolysis operation in step a) is carried out in the presence of water, preferably with the biomass (i.e., the by-product of edible flour processing containing starch) kept in an aqueous suspension.

[0055] According to a preferred aspect, this is carried out by feeding said biomass at least 5%, preferably at least 10%, and more preferably at least 20% of the initial dry weight relative to the volume of the aqueous suspension.

[0056] According to another preferred aspect, the operation is carried out by gradually feeding the biomass (e.g., semi-continuous or continuous) to maintain the solids content (by dry weight) in the hydrolysis reactor at, for example, between 5% and 50%, preferably 40% by weight relative to the volume of the aqueous suspension, and more preferably between 7% and 20% by weight relative to the volume of the aqueous suspension.

[0057] During the hydrolysis reaction, the pH value is selected based on the enzyme used, for example by adding an inorganic acid such as sulfuric acid, and the temperature. Depending on specific requirements, the pH and temperature conditions can be kept constant or adjusted during the process.

[0058] For example, according to a preferred embodiment, the reaction is carried out in the presence of amylase, the pH is advantageously maintained between 3.5 and 7 (more preferably between 4.5 and 5.5), and the temperature during the reaction is preferably maintained between 30°C and 130°C, more preferably between 40°C and 70°C, and even more preferably between 50°C and 60°C.

[0059] The duration of the hydrolysis reaction varies depending on the conditions used, particularly the type and concentration of the enzyme. Advantageously, it is 0.1 to 120 hours, for example, 0.5 to 48 hours, and more advantageously, it is 1 to 6 hours.

[0060] The hydrolysis step is optionally carried out in the presence of an antimicrobial agent and / or bactericide that inhibits the growth of contaminating microorganisms. Examples are short-chain fatty acids such as nonanoic acid, hydroxy acids (such as lactic acid and citric acid), parabens, benzalkonium chloride, and quaternary ammonium salts. Preferred examples are nonanoic acid, lactic acid, and citric acid.

[0061] Alternatively, the hydrolysis in step a) can be achieved by chemical and / or physical methods, such as using inorganic acids (e.g., HCl and H2SO4) or solid acids (e.g., sulfonated organic resins), even without enzymes.

[0062] The hydrolysis in step a) can be carried out by a continuous or semi-continuous method, or alternatively by a batch mode.

[0063] During step b) of the method, the mixture obtained at the end of the hydrolysis step is subjected to at least one solid / liquid separation operation, during which the liquid fraction containing sugars and soluble components is separated from the solid fraction containing insoluble components (including fiber, protein, and / or oligopeptides). Advantageously, one or more separation operations can be performed in series.

[0064] The separation operation in step b) is performed in accordance with techniques known to those skilled in the art, such as one or more operations selected from pressing, settling, precipitation, centrifugation, filtration or any other suitable solid-liquid separation techniques and combinations thereof.

[0065] The separation operation in step b) can therefore be performed by one or more devices capable of separating (optionally by compression) the solid fraction and the liquid fraction. Examples of suitable devices are decanters, sedimentation tanks, filter presses, hydrocyclones, belt filters, rotary filters, and centrifuges.

[0066] In addition, activated carbon and diatomaceous earth can be optionally used to assist in the separation.

[0067] According to a preferred aspect of the invention, the separation of liquid fractions is carried out by a belt filter, which is advantageously equipped with a backwashing system.

[0068] On the other hand, the separation of liquid fractions is carried out by decanter.

[0069] One or more aliquots of the liquid fraction separated by solid / liquid separation in step b) can be easily recycled back to step a), which has the dual advantages of recovering important fractions of still active enzymes and increasing the sugar concentration in the liquid phase, thus offering considerable advantages in terms of both water consumption and equipment size.

[0070] In a preferred embodiment of the method according to the invention, in step b), the mixture is first subjected to separation of insoluble solids and liquid fractions. The resulting liquid fraction can then be processed again by a further solid-liquid separation operation to recover more solid fractions, which can be combined with the former.

[0071] The separation operation described in step b) may optionally be followed by washing the solid fraction of the hydrolyzed mixture with one or more solvents to facilitate the recovery of soluble monosaccharides and simultaneously remove impurities (e.g., salts, soluble proteins, or oligopeptides). This washing step may advantageously be carried out with water, more advantageously by performing several successive washes in series, and even more advantageously by countercurrent washing and / or backwashing. The wash water may be collected, optionally concentrated, and combined with the previously separated liquid fraction.

[0072] The solid fraction obtained at the end of step b) consists primarily of insoluble substances, including insoluble fiber, protein, and / or oligopeptides, and may further contain, for example, oil and fatty acids. Preferably, the water content is less than 85%, more preferably less than 80%, and even more preferably less than 75%. The extremely low starch and ash content (starch, if present, preferably less than 5% by weight relative to dry weight, more preferably less than or equal to 3% by weight, less than or equal to 2% by weight, and even more preferably less than or equal to 1% by weight; ash content preferably less than 5% by weight relative to dry weight, more preferably less than or equal to 3% by weight) makes the solid fraction particularly suitable for use in the food industry.

[0073] Starch content can be measured using a method developed by the Laboratory for Analytical Procedures (LAP) of the National Renewable Energy Laboratory. Determination of Cellulosic Glucan Content in Starch Containing Feedstocks The determination was performed using the method described in (Michel, K.; Sluiter, J.; Payne C.; Ness, R.; Thornton, B.; Reed, M.; Schwartz, A.; and Wolfrum, E.; Technical Report NREL / TP-2800-76724, 2021).

[0074] Ash (e.g., silicates, chlorides, bromides, nitrates, sulfates, phosphates, sodium, potassium) can be removed using methods developed, for example, by the Analytical Procedures Laboratory (LAP) of the National Renewable Energy Laboratory. Determination of Ash in Biomass"(Sluiter, A.; Hames, B.; Ruiz, R.; Scarlata, C.; Sluiter, J. and Templeton, D.; Technical Report NREL / TP-510-42622, 2008) was determined by changing the holding time at 575°C to at least 5 hours.

[0075] For example, when the method of the present invention is applied to byproducts of cereal processing such as wheat, the solid fraction advantageously comprises at least 45% by weight, preferably at least 50% by weight, of polysaccharides (consisting mainly of hemicellulose and cellulose) and at least 18% by weight, preferably at least 20% by weight, of proteins (including oligopeptides and free amino acids), relative to its dry weight.

[0076] The polysaccharide in the solid fraction is advantageously composed of hemicellulose and even more preferably arabinoxylan, comprising at least 50% by weight.

[0077] The solid fraction advantageously comprises at least 30% by weight of hemicellulose, wherein the hemicellulose is preferably composed of arabinoxylan.

[0078] Cellulose and hemicellulose, for example, can be processed using methods developed by the Analytical Procedures Laboratory (LAP) of the National Renewable Energy Laboratory. Determination of Structural Carbohydrates and Lignin in Biomass Quantitative analysis was performed (Sluiter, A.; Ruiz, R.; Scarlata, C.; Sluiter, J.; Templeton, D.; Crocker, D: Technical Report NREL / TP-510-42618, 2012). Specifically, the cellulose content was determined by subtracting the starch content from the measured total dextran.

[0079] The total protein content in the solid fraction was determined by analyzing the total nitrogen content using the Kjeldahl method and then multiplying the obtained value by a conversion factor defined according to the properties of the biomass being analyzed.

[0080] Therefore, the present invention also relates to a composition prepared from byproducts of edible flour processing derived from cereals, pseudo-cereals and / or legumes, the composition being substantially starch-free and containing, relative to the dry weight of the composition, at least 45% by weight of polysaccharides (of which at least 50% by weight is hemicellulose, preferably composed of arabinoxylan), at least 18% by weight of protein (including oligopeptides and free amino acids), and preferably less than 5% by weight of ash, more preferably less than or equal to 3% by weight.

[0081] According to a preferred aspect of the method, the solid fraction is recovered and advantageously subjected to one or more subsequent concentration and / or drying steps.

[0082] This concentration and / or drying may involve one or more liquid removal stages, such as using a press, like a screw press, which has the dual advantage of increasing the recovery rate of the liquid phase and reducing the volume of the solid fraction undergoing subsequent processing.

[0083] This drying process is advantageously carried out using technologies known in the industry. The operation can be carried out intermittently or continuously, for example using plate dryers, drum dryers, fixed bed dryers, tunnel dryers, belt dryers, fluidized bed dryers, flash dryers, annular dryers, disc dryers, and sputtered bed dryers, with direct or indirect heating, and the drying gas flowing or circulating in a single cycle.

[0084] Conveniently, this operation is carried out by selecting a drying system that does not damage the solid while preserving the structure of the protein components. Systems that achieve drying by minimizing the time spent at high temperatures and maintaining relatively low temperatures (e.g., ring dryers, flash dryers) are particularly advantageous. This drying is followed advantageously by milling and separation operations (e.g., sieving, air fractionation) to facilitate the separation of the components.

[0085] A particularly advantageous aspect of the invention is the possibility of further valuing the solid fraction obtained in step b) of the method. For example, since the solid fraction is substantially starch-free, it can be easily separated into protein-rich compositions and fiber-rich compositions, both of which have high nutritional value and can be used as food additives for both human and animal nutrition. The protein component can also be used in agriculture, for example, as a biostimulant after hydrolysis.

[0086] The polysaccharide fibers (e.g., cellulose, hemicellulose) present in the solid fraction obtained from step b) of this method have the advantage of being readily separated by physical or chemical treatments, or combinations thereof, known to those skilled in the art. For example, separation can be advantageously carried out by extraction, for instance, in an alkaline environment and / or in the presence of an organic solvent (e.g., DMSO, imidazole, alcohols such as ethanol, propanol, tert-butanol), or by hydrothermal treatment, and may also be carried out by means of, for example, ultrasound or microwaves.

[0087] The methods described above are typically carried out under conditions that would lead to the degradation of the sugars present. Therefore, an unexpected advantage of the present invention is the ease with which high-value plant fibers, such as arabinoxylan, can be recovered from the composition constituting the solid fraction (which is recovered from step b) while minimizing the possibility of loss of soluble sugars that have previously been separated in the liquid fraction. For example, according to a preferred embodiment of the invention, the method includes the following additional step:

[0088] i. The solid fraction separated in step b) is selectively separated from the cellulose by extraction in an alkaline aqueous solution (possibly in the presence of a polar organic solvent such as ethanol) and subsequent acidification; and

[0089] ii. Hydrolyze one or more of the separated polysaccharide chains to reduce their molecular weight to oligosaccharides or to break them down completely into monosaccharides.

[0090] In particular, hydrolysates rich in arabinoxylan can be obtained from hemicellulose; these hydrolysates have many positive health-promoting effects and are therefore suitable for use in the food and nutritional supplement industry for the production of food and beverages. For example, the hydrolysis reaction is advantageously carried out, for instance, by enzymes, using commercial enzymes such as xylanase and arabinosease, under conditions known to those skilled in the art.

[0091] According to an alternative aspect of this method, the solid fraction undergoes an additional step of hydrolyzing the polysaccharide chains in cellulose and / or hemicellulose directly, as described above for step a) of the method, to adapt the hydrolysis mode to the desired product. This hydrolysis also facilitates the release of proteins from the solid fraction. By subjecting the resulting hydrolysate to subsequent solid / liquid separation operations, for example according to a method selected above for step b), the recovery yields of monosaccharides and possibly soluble proteins can be more or less selectively increased.

[0092] Therefore, another object of the present invention is a method for utilizing a byproduct of edible flour processing (the byproduct comprising starch), the method comprising the following steps:

[0093] (a) The byproduct is subjected to starch hydrolysis in the presence of water and preferably one or more enzymes to obtain an aqueous mixture comprising monosaccharides, fiber, protein and / or oligopeptides and optionally salts, oils and fatty acids.

[0094] (b) The aqueous mixture is subjected to solid / liquid separation to obtain a solid fraction comprising insoluble fiber and insoluble protein and / or oligopeptides, and a liquid fraction comprising soluble monosaccharides, fiber and soluble protein and / or oligopeptides, and optionally salts; and

[0095] The solid fraction is further subjected to at least partial hydrolysis of the polysaccharide chains of cellulose and / or hemicellulose contained therein.

[0096] The further hydrolysis may be carried out on one or more polysaccharide chains of cellulose and / or hemicellulose, and advantageously, the polysaccharide chains of hemicellulose may be selectively separated from those of cellulose first, for example, as described above according to steps i. and ii. According to a preferred aspect, the further hydrolysis is preferably carried out using xylanase.

[0097] In step c), the liquid fraction from step b) is purified, which includes at least one membrane separation operation (c1).

[0098] For example, such membrane separation methods can be selected from filtration (e.g., microfiltration and / or ultrafiltration and / or nanofiltration), reverse osmosis, and electrodialysis.

[0099] Preferably, the separation in step c1) includes at least one filtration operation selected from ultrafiltration and nanofiltration.

[0100] Based on the characteristics of the liquid fractions in the separation operation performed in step c1), those skilled in the art can select the type of membrane to be used by considering the material from which the membrane is prepared, its electrochemical properties, and its porosity.

[0101] The type of membrane chosen will determine the pressure and other optimal operating conditions. Those skilled in the art will be able to assess plant conditions to ensure product yield and quality, determine whether operation should be carried out in batch or continuous mode, whether and how much dialysis filtration (i.e., diluting the retentate with water and repeating the separation operation) is required, and the concentration and species rejection rate to be achieved.

[0102] For example, the separation in step c1) can be effectively performed using both organic and inorganic membranes, the organic membrane being of natural origin (e.g., rubber, polysaccharides) or synthetic origin (e.g., polymer membranes), and the inorganic membrane being, for example, a ceramic, metal, or glass membrane.

[0103] Among organic membranes, those preferred are polyamides, polyimides, polyalkylene compounds, polyetherimides, polyarylene ethers, poly(ether ketones), polycarbonates, cellulose acetate, and their derivatives.

[0104] Specific examples of suitable organic membranes are polysulfone, polyamide, polypiperazineamide, polyethylene, polytetrafluoroethylene (PTFE), polypropylene, polyvinyl alcohol, polystyrene, polybenzimidazole (PBI), polyphenylene, polyphosphazene, polyvinylidene fluoride (PVDF), polyethersulfone (PES), polyacrylonitrile (PAN), and polyvinyl chloride (PVC).

[0105] Both isotropic (or symmetrical) membranes and anisotropic (or asymmetrical) membranes, as well as composite membranes, are applicable.

[0106] Anisotropic membranes are preferred.

[0107] Membranes can be manufactured in different configurations, such as planar, tubular, capillary, or hollow fiber. Planar membranes can be used directly in pressure filtration systems, rotary systems, or wound in helical assemblies to increase the surface area to volume ratio.

[0108] The membrane separation operation according to the present invention can be carried out in intermittent or continuous mode; depending on the specific circumstances, forward (vertical) or tangential flow filtration methods are preferred.

[0109] Membrane separation is preferably performed under tangential flow.

[0110] According to the present invention, nanofiltration is preferably performed using membranes made of materials selected from the group consisting of polysulfone, polypiperazine amide, polyamide, and polyimide.

[0111] The main purpose of step c1) is to obtain a purified sugar solution (i.e., an aqueous solution of soluble monosaccharides), and secondly to obtain soluble protein fractions.

[0112] Several membrane separation operations with different rejection values ​​can be easily combined to produce one or more of the above-mentioned flows.

[0113] A particularly advantageous setup involves the use of a first nanofiltration membrane with a retention cutoff of 600-800 Da. The resulting permeate is a purified sugar solution, while the retentate is subjected to a second ultrafiltration treatment (e.g., a retention cutoff of 2-10 kDa). This treatment separates the permeate, which contains salts and other low molecular weight compounds, from the retentate, which is a concentrated solution containing proteins and soluble fibers.

[0114] The soluble protein fraction obtained from step c1) has a high organic nitrogen content (present in the form of proteins, oligopeptides, and / or simple amino acids) and a low ash content. Therefore, it is suitable for the food industry. It advantageously contains at least 10% by weight of protein, preferably at least 15% by weight, more preferably at least 20% by weight, and even more preferably 50% by weight or more of protein by weight. Examples of proteins are albumin and globulin. For example, the soluble protein content can be determined by measuring the total nitrogen content, which can be determined according to the Kjeldahl method described above, or by a dedicated analyzer. The free amino acid content is preferably 5% by weight or less of its dry weight.

[0115] Advantageously, it also contains soluble oligosaccharides, such as xylooligosaccharides and arabinoligosaccharides, preferably at a content of at least 10% by weight of dry weight. These oligosaccharides characteristically possess prebiotic activity and are therefore suitable for use in the food and nutritional supplement industries. According to a preferred aspect, the 10% by weight consists of arabinoxylan.

[0116] A further advantage of the invention is therefore the possibility of further utilizing the soluble protein fraction obtained in step c1) as a filtration retentate, particularly as a nanofiltration retentate.

[0117] For example, it can be further processed by hydrolysis of any sugar components that may be present, or by membrane treatment to separate fractions rich in soluble arabinoxylan and / or further concentrate the protein content; it can also be conveniently dried using conventional methods, such as pre-concentration followed by spray drying.

[0118] The membrane separation operation described above allows the removal of soluble protein fractions in step c1) to obtain an aqueous solution containing sugar.

[0119] Subsequently, the resulting aqueous solution is preferably passed at least once through a bed containing one or more ion exchange resins (step c2) to remove residual impurities, including any remaining organic molecules, salts, and / or amino acids.

[0120] Based on the characteristics of the liquid solution entering step c2), those skilled in the art can select the type of resin to be used by considering the material from which the resin is prepared, its electrochemical properties, its porosity, its selectivity, stability, and its adsorption capacity.

[0121] These ion exchange resins can be anionic or cationic.

[0122] Strong cationic and anionic resins are preferred. Typical commercial products are Dowex 88 and Dowex 77. An advantageous arrangement is to pass the resin sequentially through a cationic-anionic resin, and then possibly subsequently through a series-guided cationic-anionic resin, to maximize resin utilization.

[0123] The resin treatment can be carried out in a batch operation, or more conveniently, in a continuous process, such as using a rotary table or a simulated moving bed.

[0124] The purification step c), particularly the separation cl), can remove a variety of compounds and impurities from an aqueous solution containing soluble monosaccharides, thereby obtaining a purified sugar solution (i.e. containing monosaccharides).

[0125] Once concentrated, this purified solution containing monosaccharides has a purity level suitable for use in fermentation processes to produce biochemicals such as 1,4-BDO.

[0126] Each method step according to the invention can advantageously be performed independently in an intermittent, continuous, or semi-continuous mode. According to a preferred embodiment, each step in steps a)-c) is performed in a continuous mode.

[0127] According to a preferred embodiment, the method includes an optional step c3) of concentrating the purified solution containing the monosaccharide.

[0128] The concentration step is preferably performed using one or more operations known in the industry, such as adsorption, reverse osmosis, crystallization, evaporation, and distillation. Multi-effect evaporators (possibly with mechanical or thermal recompression) and low residence time devices (such as falling film or scraped film) are particularly suitable.

[0129] In a particularly advantageous aspect, a syrup containing monosaccharides, preferably monosaccharides containing 6 carbon atoms, is obtained, which can be directly used as a carbon source for the growth of microorganisms during fermentation.

[0130] The purified solution obtained at the end of step c2) or c3) preferably has an ash content of less than 0.1 wt% relative to dry weight, more preferably from 0.005% to 0.05 wt%, and a total nitrogen content of less than 0.1 wt% relative to dry weight, more preferably from 0.005 wt% to 0.05 wt%. The conductivity at 25°C is ideally less than 25 μS / cm, which is measured, for example, using a probe suitable for reading conductivity in the range of 0.001-500 μS / cm, such as the Inlab 741-ISM (Mettler Toledo), on a purified solution with an average monosaccharide concentration of 35 g / L.

[0131] For example, the ash content can be quantified using the NREL method described above by changing the holding time at 575°C to at least 5 hours.

[0132] For example, total nitrogen in a liquid sample can be determined by using an analyzer such as a TOC-L (Shimadzu) instrument, after appropriately diluting the sample to meet calibration and adjustment limits.

[0133] Therefore, a further object of the present invention is to provide a monosaccharide composition, preferably C6, obtained as a by-product of milling cereals, pseudo-cereals and / or legumes, preferably having a total monosaccharide content of 80% by weight or more, more preferably greater than or equal to 85% by weight (relative to the dry weight of the composition), an ash content of less than 0.1% by weight (relative to the dry weight), preferably from 0.005% by weight to 0.05% by weight (relative to the dry weight), and a total nitrogen content preferably less than 0.1% by weight (relative to the dry weight), preferably from 0.005% by weight, more preferably from 0.01% by weight to 0.05% by weight (relative to the dry weight).

[0134] The composition preferably contains up to 19% by weight, more preferably up to 15% by weight, and even more preferably up to 10% by weight (relative to dry matter content) of oligosaccharides, advantageously in the form of disaccharides or trisaccharides. More preferably, the composition preferably contains up to 3.0% by weight, more preferably up to 2.5% by weight, and even more preferably up to 2.0% by weight of disaccharides or trisaccharides (relative to dry matter content).

[0135] The composition has a kinematic viscosity of 30 cSt or less, preferably 26 cSt or less, and advantageously 10 to 25 cSt, when the monosaccharide concentration is 700 g / L. This composition can be obtained by the method described in this application, for example by feeding milling byproducts of cereals, pseudo-cereals, and / or legumes.

[0136] The glucose:fructose ratio in this composition is preferably 4:1 or higher, more preferably 5:1 or higher, and even more preferably 6:1 or higher.

[0137] According to a preferred aspect, the monosaccharide composition has a glucose content greater than 70% by weight and a fructose content of 10-15% by weight (relative to the dry weight of the composition); arabinose and / or xylose are preferably present. The composition can be obtained using byproducts of cereal milling (e.g., wheat) as starting biomass, as described in this application.

[0138] Depending on the needs of the final application, the fructose in the resulting syrup can be converted into glucose using techniques known to those skilled in the art.

[0139] The present invention also relates to the use of the monosaccharide composition obtained from the method and the use of the above-mentioned monosaccharide composition as a carbon source for growing microbial strains capable of producing chemical intermediates and / or polyhydroxyalkanoates.

[0140] The composition can be used as is, or after one or more further processing, the purpose of which is, for example, to concentrate the monosaccharides. It can also be used as the sole carbon source or as a mixture with other first-generation sugars. In the case of mixtures, these can contain 1 to 99% by weight of monosaccharides, preferably 10 to 80% by weight, more preferably 15 to 65% by weight of monosaccharides from the above-described monosaccharide composition.

[0141] A preferred example is its use as a carbon source for growing microbial strains capable of producing 1,4-butanediol (1,4-BDO). According to a preferred form of use, the purified monosaccharide composition described above is used as a component of the culture medium during fermentation processes in the presence of one or more microorganisms having at least one metabolic pathway for synthesizing 1,4-BDO.

[0142] For example, the fermentation can be carried out as a further method step according to the invention using the purified solution obtained in step c2) or optionally step c3).

[0143] The sugars supplied to the microorganisms for the production of 1,4-BDO can be entirely second-generation sugars produced by the saccharification of structural carbohydrates present in byproducts of edible flour processing, or a mixture of these sugars and first-generation sugars, characterized by a high level of purity. In the case of a mixture, these can contain 1% to 99% by weight, preferably 15% to 65% by weight, of sugars derived from the saccharification of byproducts of edible flour processing, relative to the total sugar content.

[0144] The culture medium may contain other substances necessary for the growth and maintenance of microorganisms during the fermentation stage, such as elements like C, H, O, N, K, S, P, Fe, Ca, Co, Mn, and Mg. Typically, the culture medium may contain one or more components selected from sugars other than glucose, protein hydrolysates, proteins, amino acids, organic acids, vitamins, mineral salts, yeast extracts, and trace elements such as cobalt, calcium, and copper. For example, cobalt, calcium, and copper can be added to the culture medium as salts such as cobalt chloride, calcium chloride, and copper chloride. Typically, the culture medium contains at least one sugar, usually glucose, and optionally one or more sugars other than glucose, at a concentration between 10 and 100 g / L. Since microorganisms consume one or more sugars during the fermentation stage, it may be necessary to reintroduce these sugars into the fermentation reactor. This reintroduction can be continuous or discontinuous, in a manner known to those skilled in the art.

[0145] To limit the amount of unutilized sugar and thus optimize the economics of the process, the supply of one or more sugars can be effectively interrupted or gradually reduced before the end of fermentation. As for the other components of the culture medium, it typically contains salts, essential minerals, and antifoaming agents. The culture medium can be prepared using any method known to those skilled in the art, such as mixing all components together or pre-mixing some of them; for example, sugars can be added later. Commercially available culture media can also be used as a starting point, with their composition appropriately modified later, for example, when the medium is contacted with microorganisms possessing at least one metabolic pathway for synthesizing 1,4-BDO from a renewable resource. During fermentation, the assembly consisting of the microorganisms and the sugar-containing culture medium is maintained under suitable conditions to utilize the metabolic pathway for the synthesis of 1,4-BDO from the renewable resource. Those skilled in the art can also monitor the process progress during fermentation, for example, by checking one or more parameters and taking measures to restore the process to conditions suitable for the production of 1,4-BDO.

[0146] The method of the present invention will now be described by way of non-limiting embodiments.

[0147] Example

[0148] Determination of cellulose and hemicellulose content

[0149] The method was developed by the Analytical Procedures Laboratory (LAP) of the National Renewable Energy Laboratory (Sluiter, A.; Ruiz, R.; Scarlata, C.; Sluiter, J.; Templeton, D.; Crocker, D.). Determination of Structural Carbohydrates and Lignin in Biomass. Technical Report NREL / TP-510-42618 (2012) was used to determine cellulose and hemicellulose.

[0150] Specifically, the cellulose content is determined by subtracting the starch content from the measured total glucan content.

[0151] Determination of starch content

[0152] Using the National Renewable Energy Laboratory's Analytical Procedures Laboratory (LAP) (Katie Michel, Justin Sluiter, Courtney Payne, Ryan Ness, Brittany Thornton, Michelle Reed, Alexa Schwartz, and Ed Wolfrum): Determination of Cellulosic Glucan Content in Starch Containing Feedstocks Technical Report NREL / TP-2800-76724, February 2021: A method for the quantitative analysis of starch was developed.

[0153] Determination of ash content

[0154] The method developed using the Analytical Procedures Laboratory (LAP) of the National Renewable Energy Laboratory (NREL) Determination of Ash in Biomass Laboratory Analytical Procedure (LAP) Publication Date: 7 / 17 / 2005 (A. Sluiter, B. Hames, R. Ruiz, C. Scarlata, J. Sluiter, and D. Templeton) Quantitatively analyzes ash content by changing the holding time at 575°C to at least 5 hours.

[0155] Protein content determination

[0156] Soluble protein in liquid samples was determined using a TOC-L instrument (Shimadzu) by appropriately diluting the sample to meet calibration and adjustment limits and multiplying the obtained value by a cereal conversion factor, specifically 5.7.

[0157] The total protein content in solid samples was determined by analyzing the total nitrogen content using the Kjeldahl method and multiplying the resulting value by the same conversion factor defined according to the properties of the biomass being analyzed.

[0158] Then, the insoluble protein is determined by the difference between total protein and soluble protein.

[0159] Example 1

[0160] By-products from the processing of edible wheat flour are categorized as follows: Figure 1 The method shown in step a) involves processing.

[0161] The water content of this by-product is 9.7 ± 0.4% by weight, and its composition is shown in Table 1.

[0162]

[0163] The intermittent enzymatic hydrolysis step a) to convert the present starch into glucose was carried out by adding water to the biomass until the dry biomass content was 16.5% w / w. Both α-amylase 'Spezyme Alpha PF' (5 μL / g dry biomass) and glucosylamylase 'Optidex L-400' (5 μL / g dry biomass) from supplier IFF were added to the mixture. Nonanoic acid was added at a rate of 0.45 g / g dry biomass, resulting in a final slurry with a total weight of 17.9 kg.

[0164] The pH was maintained at 5.0 by adding sulfuric acid, and the temperature was kept at 57°C for 6 hours.

[0165] The solid / liquid separation step b) was performed at 20°C for 10 minutes at 7500 rpm using a centrifuge equipped with a fixed-angle rotor.

[0166] The liquid filtration fraction of the recovered hydrolysate was filtered using a 25μm metal sieve.

[0167] Subsequently, the wet solids were resuspended in water at a wash water / dry hydrolysate weight ratio of 4, and finally centrifuged under the same conditions as described above. The liquid fraction from the wash was filtered as described above, and all liquid fractions were collected and sent to step c).

[0168] The solid fraction, mainly composed of insoluble fiber and protein, was recovered and dried in an oven at 50°C.

[0169] The recovered solid fraction was dried to a moisture content of 8.6 ± 0.3%, and its composition is shown in Table 2.

[0170]

[0171] A single module with a cutoff value of 600-800 Da and a diameter of 2.79 m was used. 2 A polyamide spiral membrane was used to purify a liquid fraction containing glucose and other soluble substances via nanofiltration (step c1). During filtration, the retentate was recycled and mixed with the feed stream, while the permeate was recovered separately (intermittent configuration). Furthermore, to maximize glucose recovery, a two-step dialysis filtration was performed, with a total water weight / retentate filtration weight ratio of 2.

[0172] The dialysis filtrate retained represents the soluble protein fraction, with a water content of 94.3 ± 0.1 wt%. Characterization showed that it contained a large amount of organic nitrogen (24.8 ± 0.5 wt%, relative to dry weight) in the form of amino acids and / or proteins and / or oligopeptides, a glucose content of 27.9 ± 0.2% and a fructose content of 3.6 ± 0.2 wt%, relative to dry weight).

[0173] Glucose and fructose contents were determined by high performance liquid chromatography (HPLC) with a RID detector and a Biorad Aminex HPX-87H 300 mm x 7.8 mm column and a pretreatment column (flow rate: 0.6 mL / min; oven temperature: 50 °C; detector temperature: 35 °C; eluent: 5 mM H2SO4).

[0174] Example 2

[0175] By-products from the processing of edible wheat flour are categorized as follows: Figure 1 The method shown in step a) involves processing.

[0176] The water content of this by-product is 11.3 ± 0.5% by weight, and its composition is shown in Table 3.

[0177]

[0178] The intermittent enzymatic hydrolysis step a) to convert the present starch into glucose was carried out by adding water to the biomass until the dry biomass content was 16.5% w / w. Both α-amylase 'Spezyme Alpha PF' (5 μL / g dry biomass) and glucoamylase 'Optidex L-400' (5 μL / g dry biomass) from supplier IFF were added to the mixture. The final slurry had a total weight of 350.8 kg.

[0179] The pH was maintained at 5.0 by adding sulfuric acid, and the temperature was kept at 57°C for 3 hours.

[0180] Solid / liquid separation step b) is performed using a belt filter equipped with a washing system to facilitate sugar recovery. This belt filter is a horizontal vacuum system using polypropylene filter cloth with an air permeability of 1850 L / sec / m. 2 The liquid fraction of the hydrolysate is separated in the first zone of the belt filter. Subsequently, the wet solids are washed countercurrently in three zones, with a wash water / dry hydrolysate weight ratio of 4 at the solid / liquid separation system inlet. Finally, the wet solids undergo a drying process in the last zone. The liquid fraction from the washing and drying zones is mixed with the liquid fraction from the first zone and fed into step c).

[0181] The solid fraction mainly consists of insoluble fiber and protein, which is recovered and dried by flash drying.

[0182] The recovered solid fraction was dried to a moisture content of 7.5 ± 0.4%, and its composition is shown in Table 4.

[0183]

[0184] The liquid fraction containing glucose and other soluble substances was purified by nanofiltration (step c1) using two 8.1m... 2 The module uses a polyamide spiral membrane with a filter cutoff of 600-800 Da, connected in series (total filtration area is 16.2 m²). 2 Filtration is performed in an intermittent configuration, which allows the retained material to be recycled and mixed with the feed stream, and the permeate to be recovered.

[0185] After the filtration step, the resulting retentate is subjected to four-stage dialysis filtration, so that the weight ratio of dialysis water to retentate is 4, in order to maximize glucose recovery.

[0186] The dialysis filtrate retained represents the soluble protein fraction, with a water content of 98.4 ± 0.1 wt%. Characterization showed that it contained a large amount of organic nitrogen (73.5 ± 0.1 wt%, relative to dry weight, glucose content of 10.3 ± 0.2%, and fructose content of 2.1 ± 0.2 wt%, relative to dry weight) in the form of amino acids and / or proteins and / or oligopeptides.

[0187] Glucose and fructose contents were determined by high performance liquid chromatography (HPLC) with a RID detector and a Biorad Aminex HPX-87H 300 mm x 7.8 mm column and a pretreatment column (flow rate: 0.6 mL / min; oven temperature: 50 °C; detector temperature: 35 °C; eluent: 5 mM H2SO4).

[0188] Example 3

[0189] By-products from the processing of edible wheat flour are categorized as follows: Figure 1 The method shown in step a) involves processing.

[0190] The water content of this by-product is 10.0 ± 0.9% by weight, and its composition is shown in Table 5.

[0191]

[0192] The intermittent enzymatic hydrolysis step a) to convert the present starch into glucose was carried out by adding water to the biomass until the dry biomass content was 16.5% w / w. Both α-amylase 'Spezyme Alpha PF' (5 μL / g dry biomass) and glucoamylase 'Optidex L-400' (5 μL / g dry biomass) from supplier IFF were added to the mixture.

[0193] The amount of nonanoic acid added was 0.45 g / g dry biomass, and the total weight of the final slurry was 1072.9 kg.

[0194] The pH was maintained at 5.0 by adding sulfuric acid, and the temperature was kept at 57°C for 6 hours.

[0195] Solid / liquid separation step b) is performed using a belt filter equipped with a washing system to facilitate sugar recovery. This belt filter is a horizontal vacuum system using polypropylene filter cloth with an air permeability of 1850 L / sec / m. 2 The liquid fraction of the hydrolysate is separated in the first zone of the belt filter. Subsequently, the wet solids are washed countercurrently in three zones, with a wash water / dry hydrolysate weight ratio of 4 at the solid / liquid separation system inlet. Finally, the wet solids undergo a drying process in the last zone. The liquid fraction from the washing and drying zones is mixed with the liquid fraction from the first zone and fed into step c).

[0196] The solid fraction mainly consists of insoluble fiber and protein, which is recovered and dried by flash drying.

[0197] The recovered solid fraction was dried to a moisture content of 6.9 ± 0.3%, and its composition is shown in Table 6.

[0198]

[0199] The liquid fraction containing glucose and other soluble substances was purified by nanofiltration (step c1) using two 8.1m... 2 The module uses a polyamide spiral membrane with a filter cutoff of 600-800 Da, connected in series (total filtration area is 16.2 m²). 2Filtration is carried out in a continuous configuration, allowing for the recovery of retentate and permeate in separate tanks. The retentate represents the soluble protein fraction with a water content of 90.8 ± 0.1 wt%, characterized and showing a high content of organic nitrogen (24.9 ± 0.1 wt%, relative to dry weight), glucose (44.8 ± 0.2% wt%, and fructose (3.8 ± 0.2 wt%, relative to dry weight), present in the form of amino acids and / or proteins and / or oligopeptides.

[0200] Glucose and fructose contents were determined by high performance liquid chromatography (HPLC) with a RID detector and a Biorad Aminex HPX-87H 300 mm x 7.8 mm column and a pretreatment column (flow rate: 0.6 mL / min; oven temperature: 50 °C; detector temperature: 35 °C; eluent: 5 mM H2SO4).

[0201] Then, in a single step, the membrane permeate is treated with cation exchange resin Dowex 88, followed by a single step with anion exchange resin Dowex 77 (step c2).

[0202] The aqueous solution eluted from the anion exchange resin is then concentrated in a forced circulation evaporator while maintaining a vacuum so that the concentration is carried out at a temperature below 80°C.

[0203] After concentration in the liquid phase, a 63.8% (w / w) syrup was obtained, containing more than 85% (w / w) glucose and fructose in total, and having a nitrogen content of less than 0.02% (w / w) and an ash content of 0.038% (w / w), by weight. The glucose to fructose ratio was 6.4.

[0204] Therefore, the monosaccharide syrup obtained from the byproducts by the method according to the invention has been shown to be comparable to commercially available syrups made from starch in terms of nitrogen content and ash content.

[0205] This syrup was mixed with industrial-grade (first-generation) glucose at a ratio of 32% w / w (weight of second-generation glucose and fructose / weight of total sugar) and used as a carbon source in the fermentation process for the production of 1,4-BDO. The resulting sugar mixture had a glucose concentration of 669.3 g / L and a fructose concentration of 32.24 g / L.

[0206] An *E. coli* strain possessing the 1,4-BDO synthesis metabolic pathway was inoculated into a 250 mL Erlenmeyer flask containing 25 mL of Luria Bertani medium (fortified with 15 g / L first-generation glucose). The flask was then incubated overnight at 35°C with stirring at 275 rpm to obtain the pre-inoculum.

[0207] Subsequently, the aliquots of the pre-inoculum were transferred to 1000 mL Erlenmeyer flasks containing 200 mL of the second culture medium (12.78 g / L M9 basal salt; 10 g / L first-generation glucose; 1 mL / L MgSO4 1M; 1 mL / L CaCl2 0.1M; 1.25 mL / L trace elements; 0.5 mL / L streptomycin 100 mg / mL).

[0208] The flask was incubated at 35°C with the contents stirred at 275 rpm for approximately 8 hours. After this incubation period, the optical density reached an OD value (optical density measured at 600 nm) of approximately 3-4 OD, and the culture was then inoculated into a seeding fermenter with an OD of 0.016.

[0209] Approximately 18 hours later, equal portions of the seed fermentation broth were inoculated into a production fermenter containing 1 liter of medium with an OD value of 4 (KH2PO4 1.73 g / L; (NH4)2SO4 0.83 g / L; NH4H2PO4 0.96 g / L; Na2SO4 0.30 g / L; calcium citrate·4H2O 0.038 g / L; citric acid C6H8O7 0.20 g / L; MgSO4 1M (4 mL / L); trace elements 2.5 mL / L; defoamer 0.1 mL / L) and 20 g / L of first-generation glucose.

[0210] During fermentation, the mixture of the first and second generation sugars (prepared by mixing the second generation syrup obtained according to the method of the present invention with industrial-grade glucose) is gradually fed into the fermenter by a fed-batch method so that the total concentration of glucose and fructose in the culture medium is kept constant in the range of 30-60 g / L for about 30 hours after the start of fermentation, and then the concentration is gradually reduced until the glucose concentration is about 0 g / L at the end of fermentation (about 36 hours from inoculation).

[0211] The fermentation tank was maintained under the following conditions: stirring speed 700-900 rpm, air flow rate 0.4 vvm (liters of air / liters of culture medium / minute), pH 6.75, and temperature 35℃.

[0212] Fermentation broth samples were collected at different times, and the production of 1,4-BDO was evaluated by high performance liquid chromatography (HPLC).

[0213] The content of 1,4-BDO was analyzed by HPLC using a Biorad Aminex HPX-87H 300 mm x 7.8 mm column equipped with a RID detector and a corresponding pretreatment column. The operating conditions were as follows: flow rate 0.6 mL / min, oven temperature 50 °C, detector temperature 35 °C, and 5 mM H2SO4 as eluent.

[0214] Based on the collected data, the titer and productivity were determined, including:

[0215] "Titration" (g / L): The concentration of 1,4-BDO in the reaction medium at the end of the fermentation time;

[0216] "Productivity" (g / L / h): The average rate of synthesis of 1,4-BDO, calculated in titration per hour of fermentation.

[0217] Table 7 shows a comparison of the results obtained with reference results obtained under the same operating conditions, but using only industrial-grade sugar feed.

[0218]

[0219] The performance comparison of the method for producing 1,4-BDO by fermentation clearly demonstrates that using a mixture containing monosaccharide compositions produced according to the present invention as a carbon source yields results comparable to those obtained using industrial-grade glucose. These sugars do not interfere with the normal growth of microorganisms and can be efficiently converted into 1,4-butanediol, indicating that the monosaccharide compositions produced according to the present invention have high purity. Furthermore, replacing first-generation sugars with sugars derived from byproducts of edible flour processing improves the economics and sustainability of the process.

Claims

1. A method for utilizing a byproduct from the processing of starch-containing edible flour, comprising the following steps: a) Hydrolyzing the byproduct in the presence of water to obtain an aqueous mixture comprising monosaccharides, fiber, protein and / or oligopeptides; b) subject the aqueous mixture to solid / liquid separation to obtain a solid fraction containing insoluble fiber and insoluble protein and / or oligopeptides, and a liquid fraction containing soluble monosaccharides, soluble fiber and protein and / or oligopeptides, and optionally salts. c) Purifying the liquid fraction obtained in step b) includes... c1) At least one membrane separation operation to remove soluble protein fractions and obtain an aqueous solution containing soluble monosaccharides, and subsequently c2) Pass the aqueous solution through a bed containing one or more ion exchange resins at least once to obtain a purified solution containing monosaccharides.

2. The method according to claim 1, wherein the by-product of edible flour processing is biomass obtained by milling cereals, false cereals and / or legumes.

3. The method of claim 1, wherein the product is biomass comprising more than 20% by weight of total fiber excluding starch and at least 15% by weight of protein relative to the dry weight of the biomass.

4. The method according to one or more of claims 1-3, wherein the hydrolysis in step a) is carried out in the presence of the byproduct in an amount of at least 5%, preferably at least 10%, more preferably at least 20%, and preferably less than 40% by weight, relative to the volume of the aqueous mixture.

5. The method according to one or more of claims 1-4, wherein step a) is carried out in the presence of at least one amylase, preferably selected from α-amylase, glucosylamylase (or amyloglucosidase or γ-amylase) and mixtures thereof.

6. The method according to one or more of claims 1-5, wherein one or more aliquots of the liquid fraction separated in step b) are recycled to step a).

7. The method according to one or more of claims 1-6, further comprising the additional step of drying the solid fraction separated in step b).

8. The method according to any one of claims 1-7, wherein the solid fraction separated in step b) is subjected to separation of protein components from fiber-rich components.

9. The method according to one or more of claims 1-8, further comprising the additional step of hydrolyzing the cellulose and / or hemicellulose polysaccharide chains of the solid fraction separated in step b) in the presence of water and one or more enzymes.

10. The method according to one or more of claims 1-9, wherein the separation in step c1) comprises at least one ultrafiltration and / or nanofiltration operation.

11. The method according to one or more of claims 1-10, comprising step c3 of concentrating the purified solution containing monosaccharides obtained in step c2).

12. The method of claim 11, wherein the concentration is carried out by one or more operations selected from adsorption, dialysis, reverse osmosis, crystallization, evaporation and distillation.

13. A monosaccharide composition, preferably C6, having a total monosaccharide content of 80% or higher, wherein the glucose:fructose ratio is 4:1 or higher, the ash content is less than 0.1 wt%, preferably 0.005 wt% to 0.05 wt%, and the total nitrogen content is less than 0.1 wt%, preferably 0.005 wt%, more preferably 0.01 wt% to 0.05 wt%, relative to the dry weight of the composition.

14. The monosaccharide composition according to claim 13, obtained at the end of the method according to any one of claims 1-12.

15. The use of a monosaccharide composition obtained by one or more of the methods according to claims 1-12 or the monosaccharide composition according to claim 13 as a carbon source for growing microbial strains capable of producing chemical intermediates and / or polyhydroxyalkanoates.

16. The use according to the preceding claims, for growing microbial strains capable of producing 1,4-butanediol.

17. A composition, which can be obtained as a solid fraction in step b) of the method of claim 1, is substantially free of starch and contains at least 45% by weight of polysaccharides, at least 18% by weight of proteins (including oligopeptides and free amino acids), and preferably less than 5% by weight, more preferably no more than 3% by weight of ash, based on the dry weight of the composition.

18. A composition, which can be obtained as a soluble protein fraction in step c1) of the method of claim 1, comprising one or more oligosaccharides, preferably selected from xylose oligosaccharides and / or arabinose oligosaccharides, and at least 10% protein, preferably at least 15% by weight, more preferably at least 20% by weight, relative to its dry weight.

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